RNA TEMPLATED LIGATION

Abstract

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

Claims

1. A method of detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, said method comprising: a) contacting said sample with a padlock probe and allowing said probe to hybridise to the target nucleic acid molecule; b) subjecting said sample to a ligation reaction using a DNA/RNA ligase to ligate, and thereby circularise, any probe which has hybridised to the target nucleic acid molecule; c) amplifying the ligated circularised probe from step (b) by rolling circle amplification with a DNA polymerase; and d) detecting the amplification product from step (c), thereby to detect the target nucleic acid sequence; wherein the padlock probe is provided in one or more parts, each part having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule such that, optionally after a step of cleaving the hybridised probe and/or extending a 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence; and wherein said probe comprises at least one ribonucleotide at or near to a ligation site, optionally after said step of cleaving and/or extension, and the ligated circularised probe is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.

2. The method of claim 1, wherein the target nucleic acid sequence is a target RNA sequence and wherein the target nucleic acid molecule is a target RNA molecule.

3. The method of claim 1 or claim 2, wherein the probe comprises two or more parts, wherein at least two ligation sites are created.

4. The method of any one of claims 1 to 3, wherein the probe comprises the at least one ribonucleotide at or near a ligatable 3′ end at a ligation site.

5. The method of claim 4, wherein the ligatable 3′ end is created by extension of the 3′ end of the probe or a part thereof hybridised to the target nucleic acid molecule.

6. The method of any one of claims 1 to 4, wherein the probe comprises the at least one ribonucleotide at or near the 3′ end of the probe or a part thereof.

7. The method of any one of claims 1 to 6, wherein a 3′ terminal and/or 3′ penultimate nucleotide of the probe or a part thereof is a ribonucleotide.

8. The method of any one of claims 1 to 7, wherein the probe does not comprise a ribonucleotide at a 5′ ligatable end at a ligation site.

9. The method of any one of claims 1 to 8, wherein the 5′ ligatable end of the probe, or a part thereof, is created by cleavage of the probe when it is hybridised to the target molecule.

10. The method of claim 9, wherein a first target-specific target binding site is situated internally of the 5′ end of the probe or probe part and a second target-specific binding site is situated at the 3′ end of the probe or probe part, such that upon hybridisation of the probe or probe parts to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the first target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap which is removed by cleavage thereby to create a 5′ ligatable end.

11. The method of claim 10, wherein one or more nucleotides at the 3′ end of the additional sequence which forms the 5′ flap are complementary to cognate nucleotides in the target nucleic acid molecule, wherein said nucleotides are not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe or probe part.

12. The method of claim 10 or 11, wherein the additional sequence comprises one or more ribonucleotides.

13. The method of claim 12, wherein one or more nucleotides at the 3′ end of the additional sequence are ribonucleotides.

14. The method of any one of claims 10 to 13, wherein the 3′-most nucleotide of the additional sequence is a ribonucleotide.

15. The method of any one of claims 1 to 14, wherein said probe is single circularisable oligonucleotide comprising a first target-specific binding site at or internally to the 5′ end of the probe and a second target-specific binding site at the 3′ end of the probe, wherein the first and second target-specific binding sites form or constitute the 5′ and 3′ ligatable ends of the probe respectively.

16. The method of any one of claims 9 to 15, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific target binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the additional sequence is a ribonucleotide which is complementary to the cognate nucleotide in the target nucleic acid molecule but is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe, wherein said additional sequence is removed by cleavage thereby to create the 5′ ligatable end of the probe.

17. The method of any one of claims 9 to 16, wherein cleavage is performed by an enzyme having 5′ nuclease activity or by a structure-specific nuclease.

18. The method of claim 17, wherein said enzyme is a DNA polymerase enzyme selected from Thermus aquaticus, Thermus thermophilus or Thermus flavus polymerase.

19. The method of any one of claims 9 to 18, wherein cleavage is performed by a FLAP endonuclease.

20. The method of any one of claims 1 to 19, wherein the ligated probe comprises no more than 2 consecutive ribonucleotides.

21. The method of any one of claims 1 to 20, wherein the DNA polymerase is Bst polymerase or Phi29 DNA polymerase.

22. The method of any one of claims 1 to 21, wherein the ligase is T4 RNA ligase 1, T4 RNA ligase 2 or PBCV-1 DNA ligase.

23. The method of any one of claims 1 to 22, wherein the method is for the detection of a variant base in a target nucleic acid molecule.

24. The method of claim 23, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the probe is a ribonucleotide and wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein when the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the variant base the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe and said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe are not complementary to the variant base, the nucleotide at the 3′ end of the additional sequence is removed by cleavage and the nucleotide at the 3′ end of the probe is not hybridised to the target nucleic acid molecule, thereby preventing ligation.

25. The method of claim 23, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the probe is a ribonucleotide, and wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base and wherein the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe, wherein when the nucleotide at the 5′ end of the first target-specific binding site is complementary to the variant base, said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 5′ end of the first target-specific binding site is not complementary to the variant base, said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.

26. A chimeric DNA-RNA padlock probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein: (i) the probe comprises one or more parts each having at least one target specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule such that, optionally after a step of cleaving the hybridised probe and/or extending a 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create one or more ligation sites at or adjacent to the target nucleic acid sequence, wherein ligation at said ligation sites circularises the probe; (ii) the probe comprises at least one ribonucleotide at or near to a ligation site; and (iii) the probe when ligated to form a circle is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.

27. The chimeric DNA-RNA padlock probe of claim 26, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific target binding site situated at or internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, and wherein: (i) the second target specific binding site comprises one or more ribonucleotides; (ii) where the first target specific binding site is internal to the 5′ end of the probe, the probe comprises an additional sequence 5′ to the first target specific binding site, such that when the probe is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of the probe to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; and (iii) the probe when ligated to form a circle is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.

28. The chimeric DNA-RNA padlock probe of claim 26, wherein the probe comprises two or more parts, the first part being a backbone oligonucleotide comprising a first target-specific target binding site situated at or internally of its 5′ end and a second target-specific binding site situated at its 3′ end, and one or more gap oligonucleotides which each comprise a target-specific binding site complementary to and capable of hybridising to the target nucleic acid molecule in between the first and second target-specific binding sites of the backbone oligonucleotide, and wherein: (i) the second target specific binding site of the backbone oligonucleotide and/or at the target-specific binding site of at least one gap oligonucleotide comprises one or more ribonucleotides at or near the 3′ end thereof; (ii) where the first target specific binding site is internal to the 5′ end of the backbone oligonucleotide, the backbone oligonucleotide comprises an additional sequence 5′ to the first target specific binding site, such that when the backbone oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of a gap oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; (iii) one or more gap oligonucleotides optionally comprise an additional sequence 5′ to the target-specific binding site, such that when the gap oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of another gap oligonucleotide or to the 3′ end of the backbone oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; and (iv) the backbone and gap oligonucleotides when ligated form a circle which is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.

29. The probe of claim 27 or 28, wherein the probe or backbone oligonucleotide and/or one or more gap oligonucleotides comprises an additional sequence 5′ to the first target-specific binding site or to a target-binding site of a gap oligonucleotide, wherein the nucleotide at the 3′ end of any additional sequence which forms a 5′ flap is complementary to the same cognate nucleotide in the target nucleic acid molecule as the nucleotide at the 3′ end of the probe, or at the 3′ end of the backbone and/or one or more gap oligonucleotides.

30. The probe of claim 29, wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein: (i) the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides and the nucleotide at the 3′ end of the additional sequence are complementary to a variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or (ii) the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides are not complementary to the variant base, such that the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides is not capable of hybridising to the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3′ end of the additional sequence may be removed by cleavage.

31. The probe of claim 29, wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein: (i) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a backbone or gap oligonucleotide is complementary to the variant base, the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or (ii) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a backbone or gap oligonucleotide is not complementary to the variant base, and said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.

32. A set of two or more probes as defined in claim 30 or claim 31, wherein each probe in said set of probes comprises a different nucleotide at a position in the 3′ end of the probe and/or backbone or gap oligonucleotide and first target binding site or additional sequence complementary to the variant base.

33. The set of probes of claim 32, comprising three or four probes, wherein each probe comprises a different nucleotide at said position.

34. A method of detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, said method comprising: a) contacting said sample with a ligatable probe and allowing said probe to hybridise to the target nucleic acid molecule; b) subjecting said sample to a ligation reaction using a DNA/RNA ligase to ligate any probe which has hybridised to the target nucleic molecule; c) amplifying the ligated probe from step (b) with a DNA polymerase; and d) detecting the amplification product from step (c), thereby to detect the target nucleic acid sequence; wherein each probe is provided in one or more parts each having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule, wherein one target-specific binding site of the probe or at least one target-specific binding site of a probe part is situated internally of the 5′ end of the probe or probe part such that upon hybridisation of the probe or probe part to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap, wherein one or more of the nucleotides at the 3′ end of the 5′ additional sequence is a ribonucleotide, and wherein after a step of cleaving the hybridised probe to remove the 5′ flap, and optionally after a step of extending the 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence.

35. The method of claim 34, wherein the probe comprises two or more parts, and wherein at least two ligation sites are created.

36. The method of claim 34 or 35, wherein the ligatable 3′ end is created by extension of the 3′ end of the probe or a part thereof hybridised to the target nucleic acid molecule.

37. The method of any one of claims 34 to 36, wherein one or more nucleotides at the 3′ end of the additional sequence which forms the 5′ flap are complementary to cognate nucleotides in the target nucleic acid molecule, wherein said nucleotides are not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe or probe part.

38. The method of any one of claims 34 to 37, wherein the probe is a padlock probe comprising one or more parts which are ligated together to form a circle, optionally after an extension step.

39. The method of claim 38, wherein said probe is a single circularisable oligonucleotide comprising first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site at the 3′ end of the probe, wherein the first and second target-specific binding sites form or constitute the 5′ and 3′ ligatable ends of the probe respectively.

40. The method of any one of claims 34 to 39, wherein the probe is a single circularisable oligonucleotide comprising a first target specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the nucleotide at the 3′ end of the additional sequence is complementary to the cognate nucleotide in the target nucleic acid molecule but is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe.

41. The method of any one of claims 34 to 40, wherein cleavage is performed by an enzyme having 5′ nuclease activity or by a structure-specific nuclease.

42. The method of claim 41, wherein said enzyme is a DNA polymerase enzyme selected from Thermus aquaticus, Thermus thermophilus or Thermus flavus polymerase.

43. The method of any one of claims 34 to 40, wherein cleavage is performed by a FLAP endonuclease.

44. The method of any one of claims 34 to 43, wherein the ligated probe comprises no more than 4 consecutive ribonucleotides, preferably wherein the ligated probe comprises no more than 3, or no more than 2 consecutive ribonucleotides.

45. The method of any one of claims 34 to 44, wherein the ligated probe does not comprise a ribonucleotide at or near to a ligation site.

46. The method of any one of claims 34 to 45, wherein where the probe is circularised by ligation, the amplification is rolling circle amplification.

47. The method of any one of claims 34 to 46, wherein amplification is PCR or a variant thereof, SDA, HDA, LAMP or SMAP.

48. The method of any one of claims 34 to 47, wherein the DNA polymerase is Bst polymerase or Phi29 DNA polymerase.

49. The method of any one of claims 34 to 48, wherein the ligase is T4 RNA ligase 1, T4 RNA ligase 2 or PBCV-1 DNA ligase.

50. The method of any one of claims 34 to 49, wherein the method is for the detection of a variant base in a target nucleic acid molecule.

51. The method of claim 50, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at 3′ end of the probe, wherein the nucleotide at the 3′ end of the probe and the 3′ end of the additional sequence are complementary to the same nucleotide, wherein when the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the variant base the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe and the additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe are not complementary to the variant base, the nucleotide at the 3′ end of the additional sequence is removed by cleavage and the nucleotide at the 3′ end of the probe is not hybridised to the target nucleic acid molecule, thereby preventing ligation.

52. The method of claim 51, wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base and wherein the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe, wherein when the nucleotide at the 5′ end of the first target-specific binding site is complementary to the variant base, said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 5′ end of the first target-specific binding site is not complementary to the variant base, said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.

53. A chimeric DNA-RNA probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein the probe comprises one or more parts each having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequences and hybridises thereto, wherein one target-specific binding site of the probe or at least one target-specific binding site of a probe part is situated internally of the 5′ end of the probe or probe part such that upon hybridisation of the probe or probe part to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap, wherein one or more of the nucleotides at the 3′ end of the 5′ additional sequence is a ribonucleotide, and wherein after a step of cleaving the hybridised probe to remove the 5′ flap, and optionally after a step of extending the 3′ end of the probe or a probe part using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence.

54. The chimeric DNA-RNA probe of claim 53, wherein the probe is a padlock probe, wherein the probe hybridises to the target nucleic acid molecule such that after cleavage of the additional sequence and optionally after a step of extending the 3′ end of the probe or a probe part using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create one or more ligation sites at or adjacent to the target nucleic acid molecule and wherein ligation at said ligation sites circularises the probe.

55. The chimeric DNA-RNA probe of claim 54, wherein the probe is a single circularisable oligonucleotide comprising at first target-specific target binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe.

56. The chimeric DNA-RNA probe of claim 55, wherein the probe comprises two or more parts, the first part being a backbone oligonucleotide comprising a first target-specific target binding site situated internally of its 5′ end and a second target-specific binding site situated at its 3′ end, and one or more gap oligonucleotides which each comprise a target-specific binding site complementary to and capable of hybridising to the target nucleic acid molecule in between the first and second target-specific binding sites of the backbone oligonucleotide, and wherein one or more gap oligonucleotides optionally comprise an additional sequence 5′ to the target-specific binding site, such that when the gap oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of another gap oligonucleotide or to the 3′ end of the backbone oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides.

57. The chimeric DNA-RNA probe of any one of claims 53 to 56, wherein the nucleotide at the 3′ end of any additional sequence which forms a 5′ flap is complementary to the same cognate nucleotide in the target nucleic acid molecule as the nucleotide at the 3′ end of the probe or at the 3′ end of the backbone and/or one or more gap oligonucleotides.

58. The probe of claim 57, wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein: (i) the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to a variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or probe part or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or (ii) the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are not complementary to the variant base, such that the nucleotide at the 3′ end of the probe or probe part or backbone and/or gap oligonucleotides is not capable of hybridising to the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3′ end of the additional sequence may also be removed by cleavage.

59. The probe of claim 57, wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein: (i) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a probe part or backbone or gap oligonucleotide is complementary to the variant base, the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or (ii) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a probe part or a backbone or gap oligonucleotide is not complementary to the variant base, and said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.

60. A set of two or more probes as defined in claim 58 or 59, wherein each probe in said set comprises a different nucleotide at a position in the 3′ end of the probe or probe part or backbone or gap oligonucleotide and first target binding site or additional sequence complementary to the variant base.

61. The set of probes of claim 60, comprising three or four probes, wherein each probe comprises a different nucleotide at said position.

62. A method of synthesising a DNA molecule by rolling circle amplification, said method comprising contacting a circular template nucleic acid molecule comprising at least one ribonucleotide and no more than four consecutive ribonucleotides with a Phi29 DNA polymerase, and generating a DNA molecule comprising multiple tandem repeats of the reverse complement sequence to the template nucleic acid molecule, wherein the sequence of the Phi29 DNA polymerase has not been modified to increase RT activity.

63. The method of claim 62, wherein the template nucleic acid molecule comprises no more than 3 consecutive ribonucleotides.

64. The method of claim 62 or 63, wherein the template nucleic acid molecule comprises no more than 2 consecutive ribonucleotides.

65. The method of any one of claims 62 to 64, wherein the template nucleic acid molecule comprises 2 or more consecutive ribonucleotides.

66. The method of any one of claims 62 to 65, wherein the template nucleic acid molecule comprises one or more single ribonucleotides.

67. The method of any one of claims 62 to 66, wherein the template nucleic acid molecule comprises ribonucleotides within a single site within the template nucleic acid molecule.

68. The method of any one of claims 62 to 67, wherein the template nucleic acid molecule comprises 1 ribonucleotide.

69. The method of any one of claims 62 to 66, wherein the template nucleic acid molecule contains ribonucleotides at two or more separate sites, and comprises one or more deoxyribonucleotides between each site.

70. The method of claim 69, wherein the template nucleic acid molecule contains ribonucleotides at two or more separate sites, wherein two sites are separated by a single deoxyribonucleotide.

71. The method of claim 65, wherein the consecutive ribonucleotides are homogeneous.

72. The method of any one of claims 62 to 70, wherein at least one of the ribonucleotides is a pyrimidine ribonucleotide, or the method of claim 71 wherein the consecutive ribonucleotides are pyrimidine ribonucleotides.

73. The method of claim 69 or 70, wherein each site contains ribonucleotides as defined in any one of claim 63 to 66 or 71.

74. The method of any one of claims 62 to 73, wherein the ribonucleotide is not a chemical modification, analogue or derivative of a ribonucleotide.

75. The method of any one of claims 62 to 74, wherein the circular nucleic acid molecule is formed following the ligation of a padlock probe provided in one or more parts using a target nucleic acid molecule as a template, wherein said ligation comprises ligation of ligatable ends of a probe or probe parts to create a ligation site.

76. Use of Phi29 DNA polymerase as a reverse transcriptase enzyme wherein the sequence of the Phi29 polymerase has not been modified to increase RT activity.

Description

[0221] The present invention may be better understood with reference to the Examples and Figures, in which:

[0222] FIG. 1 shows the effect of a RNA nucleotide at the 3′ ligatable end at a ligation site using PBCV-1 ligase in the ligation of a padlock probe templated by an RNA. A: Experiment overview. B: Padlock probes (PLP) targeting let-7 family members were designed with RNA or DNA terminal 3′ nucleotides. Probes were hybridised with matching templates, ligated with PBCV-1 and amplified. A total number of RCA products (RCP) for each PLP/miRNA pair is shown in the column plot. y-axis shows the number of RCPs while type of miRNA is depicted on x-axis. Error bars±s.d.; n=2.

[0223] FIG. 2 shows the effect of a RNA nucleotide at the 3′ ligatable end at a ligation site using PBCV-1 or T4Rnl2 ligase. Ligation of a 3′-OH(N)/5′-p(N) vs 3′-OH(rN)/5′-p(N) using an RNA template was compared. Full DNA and chimeric padlock probes were hybridised with a corresponding RNA target and ligated with (A) PBCV-1 and (B) T4Rnl2. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the RNA template used. Error bars±s.d.; n=2. A greater number of ligation products was seen for all target RNAs for PBCV-1, and a large increase in the number of ligation products was seen for all target RNAs for T4Rnl2 ligase when the probe comprised a 3′ ribonucleotide at its 3′ end.

[0224] FIG. 3 shows the effect of 3′-OH(rN) mismatches on nick sealing by PBCV-1 and T4Rnl2 ligase. Numbers of RCPs for each RNA template (FIGS. 3B and 3C) were added and presented as percentage within an iLock probe group (FIG. 3A) for each ligase enzyme.

[0225] FIG. 4 shows the effect of RNA substitutions at various positions in an Invader padlock (iLock) probe used in an RNA detection assay with PBCV-1 ligase. The recognition of the invader structure and structure-specific nucleolytic activity of Taq DNA polymerase can vary for different RNA substitutions. A: targeting let-7a with iLock probe, showing the arrangement of the first and second target-specific binding sits of the iLock probe, and a 5′ additional sequence. RNA nucleotides were introduced at different positions: at the terminal 3′ end (3); at the 3′-most nucleotide in the 5′ flap which competes with the terminal 3′ nucleotide at the end of the probe for target binding (displaced base, D); the base in the first target binding site that becomes the 5′ ligatable end (provides the 5′-phosphorylated donor) after cleavage to remove the additional sequence (iLock probe activation) (5); the entire flap sequence (F). B: The circularisation of six iLock designs was assessed: DNA-only iLock; an iLock with the (3) modification (iLock-3); an iLock with the (3) and (D) modifications (iLock-3D); an iLock with the (3), (D) and (5) modifications (iLock-3D5); an iLock with the (3), (D) and (F) modifications (iLock-3DF); and an iLock with the (D) and (F) modifications (iLock-DF). The total number of RCPs detected for each iLock probe is showed on x-axis. Probes comprising the (3) modification, and the (3) modification in combination with the (D) or the (D) and (F) modifications showed a large increase in the number of RCPs generated relative to the DNA-only iLock. The combination of the (3) modification with the (D) and (5) modifications, and of the (D) and (F) modifications, showed a much smaller increase in the relative number of RCPs generated relative to the DNA-only iLock. C: PAGE of the iLock DNA, iLock-3 and iLock-3D probes after activation and ligation, without (lanes 1-3) and with Taq DNA polymerase (lanes 4-6). Non-activated iLock probe (79) is shortened upon activation by 14 nt (65) and ligated (seen as the high molecular weight band at the top of the gel). Lane 4: a band for activated, unligated probe is visible (65 nt), the band for uncleaved probe is clearly visible (79 nt) and only a faint band for ligated probe is visible. Lane 5: no band for unligated probe is visible, the band for uncleaved probe is clearly visible (65 nt) and a band for ligated probe is visible. Lane 6: no band for unligated probe is visible, the band for uncleaved probe is faint (65 nt) and a strong band for ligated probe is visible. Together these data show that a ribonuclease at a 3′ ligatable end at a ligation site improves ligation (lanes 4 and 5), and that a ribonuclease at the 3′-most position in an additional sequence which is cleaved in an Invader assay improves cleavage (lanes 5 and 6).

[0226] FIG. 5 shows a comparison of chimeric and non-chimeric iLock probes ligation. A: 3D and non-chimeric iLock probes performance on longer, non-miRNA targets. Total number of RCPs for each probe on matching polymorphic templates is showed on y-axis. B: a comparison of chimeric and non-chimeric iLock probes on miR21 using PBCV-1 and T4Rnl2. Total number of RCPs for chimeric or non-chimeric iLock probes is presented on y-axis. Ligase used is depicted on the x-axis. Error bars±s.d.; n=2. Both ligases demonstrate improved ligation when a chimeric probe is used.

[0227] FIG. 6 shows chimeric iLock probe ligation efficiency and fidelity on non-miRNA templates for PBCV-1 and T4Rnl2 ligase. A and B: Fidelity of nick sealing by PBCV-1 and T4Rnl2 ligases on matching polymorphic RNA templates. C and D: Data presented in (A) and (B) but as a total number of RCPs generated for each iLock probe on each polymorphic template. Error bars±s.d.; n=2.

[0228] FIG. 7 shows the multiplexed detection of let-7 miRNA isoforms using chimeric iLock probes and PBCV-1 ligase. A: miRNA-specific barcode (NN) was embedded in the probe backbone, between anchor primer hybridisation region and a sequencing library hybridisation site. During sequencing, anchor primer (AP) hybridises to the RCP and pool of sequencing library oligonucleotides compete with each other for hybridisation based on the nucleotide at their 5′ end. Library containing terminal T was 3′-FITC labelled; G—3′Cy3 labelled; A—3′Cy5 labelled. Ligase joins a library oligonucleotide corresponding to a barcode base. B: imaging of a first barcode base by sequencing by ligation (SBL). AP: all RCPs stained with AP. Images of each barcode base is presented as well as merged image. Scale bar 5 μm. C: miRNAs were mixed in stoichiometric ratios as stated on the x-axis. 1:1:1 represents equal ratio and 0:0:0 no template control. Total number of reads is depicted on the y-axis. Error bars±s.d.; n=number of samples imaged=2. A 1:1:3 ratio generated similar number of RCPs for each template.

[0229] FIG. 8 shows a comparison of the ligation efficiencies of a padlock probe, and a chimeric padlock probe comprising 1 (R1pd) or 2 (R2pd) ribonucleotides at the 3′ end, for PBCV-1 ligase and T4 RN12 ligase at both high and low concentrations, using (A) an RNA template or (B) a DNA template. Chimeric padlock probes were shown to be ligated and amplified more efficiently than DNA-only padlock probes for both RNA and DNA targets for both ligases.

[0230] FIG. 9 shows the detection of KRAS wt and mutant RNA in situ using DNA padlock probes or chimeric padlock probes. A: microscopy image showing detection of mutant and WT RNA using padlock probes (top) or chimeric padlock probes (bottom). B: Average number of mutant RCPs and wild type RCPs per cell in both cell lines A549 and OncoDG1. The efficiency of chimeric padlock probes is much higher in both cases. The specificity is high enough to distinguish between mutant and wild type KRAS (more mutant RCPs in A549 and more wild type RCPs in OncoDG1).

[0231] FIG. 10 shows the detection of KRAS wt and mutant RNA in situ using chimeric iLock probes. A: microscopy image showing detection of mutant and WT RNA using chimeric iLock probes. B: Average number of mutant RCPs and wild type RCPs per cell in both cell lines A549 and OncoDG1. The specificity is high enough to distinguish between mutant and wild type KRAS (more mutant RCPs in A549 and more wild type RCPs in OncoDG1).

[0232] FIGS. 11A-C show detection of target RNA molecules using gap-fill polymerisation and an iLock probe. A: number of RCPs counted in solution after gap-fill polymerization with reverse transcriptase and Taq cleavage+PBCV-1 ligase ligation (all at once) followed by RCA, using chimeric iLock probes. B: in situ detection of RCPs using gap-fill polymerisation and chimeric iLock probes.

[0233] FIG. 12 shows a design for a 2-part iLock probe comprising a ribonucleotide at the end of the backbone and gap oligonucleotides. A target RNA molecule (1) is contacted with a 2-part iLock probe comprising a backbone oligonucleotide (2) and a gap-fill oligonucleotide (3). Both the backbone oligonucleotide and the gap-fill oligonucleotide comprise an additional sequence at their 5′ end which is not hybridised to the target RNA molecule (4) and a ribonucleotide at their 3′ end (5).

[0234] FIG. 13 shows the effect of RNA substitutions on rolling circle amplification with Phi29 DNA polymerase. A: Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). B: Circles with 0-7 RNA substations in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars±s.d.; n=2. The same RCA reactions with chimeric circles were also monitored in real-time measuring Sybr gold incorporation on a qPCR instrument (C and E). C: RCA reaction curves of circles with 0, 1, 2 or 3 RNA substitutes. D: RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. E: Real time data of the same RCA reactions as in B with 0-7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to see difference between the samples with low RCA efficiency, fluorescence between 4 000 and 6 000 is shown.

[0235] FIG. 14 shows that chimeric padlock probes comprising a 3′ ribonucleotide are more readily ligated using PBCV-1 ligase than a padlock probe which comprises a deoxyribonucleotide at its 3′ end. Lanes 1-6—chimeric padlock probes. Lanes 7-12—non-chimeric padlock probes. A ligated probe product is shown (*) as the heavier fragment. This is clearly visible after 1-2 minutes for chimeric probes (lanes 2-3), whereas this only becomes clearly visible at later time points for non-chimeric probes (lane 12).

[0236] FIG. 15 shows a gap-fill padlock probe (2) comprising a ribonucleotide (3) at the 3′ end of a 5′ additional sequence (4), which is cleaved prior to ligation. A 3′ end of the probe (5) hybridised to the target nucleic acid molecule (1) may be extended by gap-fill polymerisation.

[0237] FIG. 16 shows a padlock probe (2), which comprising ribonucleotides (3) at positions other than at or near to a ligation site (4) hybridised to a target nucleic acid molecule (1).

[0238] FIG. 17 shows a padlock probe (2) comprising a backbone oligonucleotide provided in two parts, and a ligation template (4) which hybridises to each part of the backbone oligonucleotide in order to template ligation. As shown, the nucleotide at the 3′ ligatable end in this ligation site is a ribonucleotide.

[0239] FIG. 18 shows the rates of amplification of circularised padlock probes comprising different ribonucleotides. A: Real-time RCA curves of circles containing 1, 2 or 3 consecutive RNA substitutions of all four RNA bases are displayed. Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulting from SybrGold incorporation into RCPs. Representative data are shown for each experiment. B: RCA rates for the positive control (pure DNA circle—bottom left), negative control (no circle) and circles with 2, 3, 5 and 7 consecutive RNA substitutions, as well as circles with RNA substitutions interspersed with DNA bases are displayed. Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions.

[0240] FIG. 19 shows that limited replication of RNA-enriched padlock probes is not recovered in the presence of M-MuLV reverse transcriptase. Amplification curves of padlock probes with 0-7 RNA substations in the backbone are displayed. Rate of RCA was monitored by measuring fluorescence build-up (y-axis; 3000-30000) resulted from SybrGold incorporation into RCPs. Replication is shown for circles without additional reverse-transcriptase (upper panel) and with additional M-MuLV reverse transcriptase (lower panel).

[0241] FIG. 20 shows stacked graphs showing incorporation of expected dNTPs during RCA reverse-transcription. RNA-containing padlock probes were amplified, monomerised and sequenced. RCA monomers were generated from the control DNA circle (upper row), and circles containing rA, rC, rG and rU at the first RNA position (R1), and rUrU, rArA, rCrC and rGrG at their R1 and R2 positions (full oligo sequences in table 9). Sequencing reads were aligned and frequency of each base in every position was calculated. Size of each base is proportional to the base frequency. Positions R1 and R2 (relative to RNA positions in the padlock probe backbone) are indicated by the box and position R1 was highlighted (see arrow).

[0242] FIG. 21 shows the in situ detection of ACTB mRNA in cultured human (BjhTERT) and mouse (MEF) fibroblasts. A: Detection of human and mouse ACTB mRNA in BjhTERT and MEF cells using chimeric and non-chimeric chimeric padlock probes (PLP) and iLock probes. Probes for both targets were included in each sample, and showed good levels of target specificity. B: Average number of RCPs per cell arising from each probe are shown for each cell line using chimeric and DNA-only PLPs and iLocks. PLP: DNA-only padlock probe; PLPr—3′-(rN) PLP; RiLock: RNA iLock; iLock: DNA-only iLock. For each probe, signal from human-specific probes is top, and signal from mouse-specific probes is bottom. In BjhTERT, human ACTB-specific PLP (top box plot) shows fewer blobs than RNA PLPr (second boxplot). Mouse-specific PLP and PLPr shows no signal. For iLock (3.sup.rd and 4.sup.th box plots) RiLocks show a higher median than iLocks (boxplot is a bit shifted) but signal amount is much lower comparing to PLPs. Corresponding data was obtained for MEF mouse cells, with signals from mouse-specific probes higher than for human-specific probes.

[0243] FIG. 22 shows in situ detection of miR21 RNA immobilised on a solid surface. A: miR21 was immobilised and complementary probe (labelled with fluorescent dye) was hybridised. Edge of the silicone chamber was imaged intentionally, to visualise the immobilisation effect; B) When miR21 was not added, complementary probe generated no visible fluorescence; detection of mir21 with non-chimeric PLPs (C) chimeric PLPs (E) iLock probes (D) and chimeric iLock probes (F). Number of RCPs quantified is presented as total number of RCP/field of view (FOV).

[0244] FIG. 23 shows in situ multiplexed RNA detection using chimeric padlock probes and in situ sequencing in mouse brain tissue sections. Upper panel shows an overview image of the mouse brain tissue section with nuclei stained in DAPI and the anchor probe-stained RCA products generated from chimeric PLPs targeting 18 different neuronal genes (with 5 probes per gene each=90 different probes in total). Below, an area of the left overview image where individual cells are visible.

[0245] FIGS. 24A-D show Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. (FIGS. 24A-B) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg.sup.2+ and Mn.sup.2+ (solid and dashed lines respectively). (FIG. 24C) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg.sup.2+ (solid lines) and Mn.sup.2+ (dashed lines). (FIG. 24D) RCA for the control PLP (non-chimeric DNA circle, with Mg.sup.2+ (solid) and Mn.sup.2+ (dashed line) are displayed.

[0246] FIG. 25 shows RCA of chimeric circular substrates with RNA substitutions organised in different patterns. Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SybrGold incorporation into RCPs. 4, 5 and 7 consecutive substitution (yellow, red, green); 3, 6 RNA substitutions interspaced with 1 or 2 DNA bases (blue, grey) as well as 3 and 8 RNA substitutions interspaced with larger number of DNA bases (orange, magenta) were introduced in the PLP backbone as indicated in the legend (only fragment of a backbone fragment is depicted, full PLP sequences as indicated. Averaged data from a duplicated experiment. RCA was conducted in the presence of magnesium and manganese ions (solid and dashed lines respectively).

[0247] FIG. 26 shows the effect of RNA substitutions on 3′-OH(rG) and 3′-OH(G) padlock probe stability and ligation with PBCV-1 ligase on RNA. A: PBCV-1 ligase titration. Total number of RCPs (y-axis) generated for each ligase concentration (x axis) during 30 min ligation. For each time point, data for chimeric probes are shown on the left and non-chimeric probes are show on the right. B: To evaluate stability of chimeric padlock probes during first minutes of the reaction 26.5 nM (62 mU/μL) concentration was used. Ligation reaction was stopped by heat inactivating enzyme at 70° C. for 10 min. Total number of RCA products (y-axis) for given time point (x-axis) is presented.

[0248] FIG. 27 shows a comparison of chimeric and non-chimeric iLock probes ligation on miR21 and let-7f using PBCV-1 and T4Rnl2. Total number of RCPs for chimeric (left) or non-chimeric (right). iLock probes is presented on y-axis with PBCV-1 or T4Rnl2 (x-axis). Data is presented for miR21 and let-7f RNA template.

[0249] FIG. 28 shows the effect of 3′-OH(rN) mismatches on iLock activation and nick sealing fidelity for PBCV-1 and T4Rnl2 ligase. A and B: Chimeric iLock-3D (left) and non-chimeric (right) iLock probes performance on polymorphic RNA targets. Total number of RCPs for each probe on matching polymorphic templates is showed on y-axis for A) PBCV-1 DNA ligase and B) T4Rnl2. NC—negative control. C: Fidelity of nick sealing by PBCV-1 DNA ligase (left panel) and T4Rnl2 (right panel) using 3D-type iLock probes on RNA. Numbers of RCPs for the same iLock probe on each RNA template were added and presented as percentage within an iLock probe group. Calculated proportion for the expected probe pair is highlighted.

[0250] FIG. 29 shows PBCV-1 and T4Rnl2 ligase chimeric iLock probes ligation efficiency and fidelity on polymorphic templates. Total number of RCPs generated and quantified (y-axis) for each iLock probe on each polymorphic template is shown in FIG. 28 for A) PBCV-1 DNA ligase and B) T4Rnl2.

[0251] FIG. 30 shows dNTPs incorporation during RCA reverse-transcription. Padlock probes were monomerised, amplified and sequenced, as described in the Examples. Samples with single and double RNA bases were sequenced with this approach as depicted next to individual graphs. Sequencing reads were aligned and frequency of each base in every position was calculated. Size of each base is proportional to the base frequency. RCA reaction was conducted in the presence of magnesium and manganese ions as indicated above graphs. Position of RNA bases is indicated with the box. DNA/RNA sequence present originally in the PLP sequence is depicted on right-hand side.

[0252] FIG. 31 shows the misincorporation rate for every position in the padlock probe backbone during RCA reverse transcription. Probability of misincorporation of unexpected nucleotide at every position for padlock probes with single- (left hand plots) and double-RNA substitutions (right hand plots) were calculated as Incorporation error [%]=1-number of reads with expected nucleotide/total number of reads. Average error (y-axis) is shown for each base of the sequenced read (x-axis) and for every sample analysed. RCA reaction was conducted in the presence of manganese (A) and magnesium (B).

[0253] FIG. 32 shows the effect of RNA substitutions in circular templates on rolling circle amplification with phi29DNA polymerase. (A) Circles with 0-7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars±S.D.; n=2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument (B and C). (B) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. (C) Real-time data of the same RCA reactions as in B with 0-7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

[0254] FIG. 33 shows DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. (A) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Illumina adapter sequences. PCR products were extended using Illumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. (B) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1=dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. (C) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%]=1−number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single-(upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

EXAMPLES

Example 1—Detection of miRNAs Using Chimeric DNA/RNA iLock Probes Utilizing Novel Activity of PBCV-1 DNA Ligase: RNA-Templated Ligation of ssRNA

[0255] Material and Methods

[0256] Oligonucleotides Used in the Study

[0257] All oligonucleotides used were purchased from IDT (Integrated DNA Technologies, Inc., Coralville, Iowa, USA) using following synthesis and purification conditions: DNA padlock probes and iLock probes: 4 nM of standard desalted Ultramer® DNA oligonucleotides; chimeric padlock and iLock probes: 4 nM of standard desalted Ultramer® RNA oligonucleotides; decorator probes: HPLC purified DNA oligonucleotides with 5′ conjugated fluorophore. All padlock probes were pre-phosphorylated on the 5′ terminus to permit ligation. RNA templates harbouring centrally located polymorphic site are shown in Table 1 (benchmark oligonucleotides) with the polymorphism indicated with an asterisk.

[0258] Padlock probes were designed, such that terminal arms would form a nicked circle when base paired with attended RNA targets and discriminatory base was localized at the 3′ terminus of the probe (table 1). miRNA padlock probes used in this study are shown in table 1. Chimeric padlock probes were ordered with a terminal 3′-OH RNA. iLock probes were used in the present work for comparative purposes (table 2). Standardized chimeric iLock probes design includes RNA substitution on the terminal 3′ base as well as a base in the 5′ arm that 3′ terminal base was competing for target binding with (displaced base, FIG. 4A, table 2). Two types of probe barcoding methods were used: traditional and compatible with sequencing-by-ligation read-out (used in chimeric, miRNA targeting iLock probes). For traditional rolling circle product (RCP) staining and digital quantitation, a reporter sequence was embedded in the sequence linking the probe arms, separated from probe arms with series of 10 adenines (table 2). For the latter, a consensus backbone with unique probe-specific barcode was used (table 3). To allow barcode decoding, common anchoring primer sequence was embedded in the probe backbone, followed by two-bases barcode and a sequencing library anchoring sequence (table 3).

[0259] RNA Detection Assay and Digital Quantitation of Amplified iLock and Padlock Probes

[0260] iLock activation (cleavage) was performed in 4:1 probe to template excess (typically, 2 nM iLock probe was mixed with 0.5 nM RNA template). Duplicate reactions were incubated in a heated-lid thermocycler at 51° C. for 30 min, in a 10 μL volume containing 1 U of Taq DNA polymerase (ThermoFisher Scientific), 4 U RNaseiInhibitor and 1×Taq polymerase buffer supplied with 8 mM MgCl2. Next, 3 μL of sample volume was transferred to a ligation reaction mix supplemented with 3.75 U of PBCV-1 DNA ligase (SplintR, M0375S, NEB) or 4 U of T4Rnl2 (M0239S, NEB) in respective buffers in a final volume of 15 μL. The reactions were incubated at 37° C. for 30 min. For padlock probes, identical ligation conditions were applied, excluding the activation step. For RCA, 5 μL of the ligation reaction was incubated with 10 nmole decorator probe, 0.125 mM dNTPs, 0.2 μg/μl BSA, 250 mU of Phi29 Polymerase (Monserate Biotechnology Group) and 1× phi29 reaction buffer (Thermo Fisher) in a final volume of 25 μl at 37° C. for 60 minutes. The polymerase was heat inactivated at 65° C. for 3 minutes and allowed to cool to room temperature. Estimated final concentration of amplified products was 5 pM and 20 pM for padlock and iLock probes respectively, unless stated otherwise. 15 μl of the RCA sample were analysed using the Aquila 400 Detection Unit (Q-linea, Uppsala). If RCPs concentration was outside the instrument's dynamic range, samples were diluted in 4 nM of the decorator probe in 1× labelling solution (20 mM EDTA, 20 mM Tris-HCl (pH 7.5), 0.05% Tween 20 and 1 M NaCl), incubated at 65° C. for 3 min, allowed to cool at room temperature for 15 min and recounted. Template-negative reactions were run in parallel with every experiment as a control.

[0261] Multiplexed miRNA Detection Using Chimeric iLock Probes and Sequencing-by Ligation

[0262] To test if chimeric iLock probes could be used to detect miRNA expression variation in RNA mixtures, we have combined (let-7f):(let-7e):(let-7d) miRNAs in (1):(1):(1), (3):(1):(1), (1):(3):(1) and (1):(1):(3) ratios. Baseline miRNA concentration during iLock activation step was 0.5 nM and 1.5 nM for samples where miRNA concentration was increased. 2 nM cocktail of let-7f, let-7e, let-7d chimeric iLock probes was used, each embedded with a unique two-nucleotide barcode (table 3) and sequencing-by-ligation chemistry sequences. Protocol was conducted as described above (using PBCV-1 ligase) except that 10 μL droplets of RCA products were spotted on positively charged microscope slides (Superfrost Plus, Menzel Glaser) and evaporated at 55° C. for 10 min. 50 μL volume silicone chamber (Secure-Seal hybridization chamber, Sigma) was mounted over each droplet and samples were washed 3× with 1×TBS. 0.5 μM anchor primer was hybridised in 2×SSC, 20% formamide at room temperature for 30 min. Followed by 3× washes with 1×TBS, RCPs were mixed with sequencing mixture, containing 1×T4 DNA Ligase buffer, 10 μg BSA, 1 mM ATP, 0.1 μM sequencing oligonucleotides (table 3) and 5 U of T4 DNA Ligase. Slides were incubated at room temperature for 60 min. After 3× washes with 1×TBS, silicone chambers were removed, slides rinsed with 100% ETOH, air-dried and mounted (SlowFade antifade, ThermoFisher). Images of RCPs were acquired using 20× objective.

[0263] To visualise activation and ligation efficiency of various chimeric iLock probes (table 2) products were separated electrophoretically. 5 μM synthetic RNA and 2.5 μM probe were processed as above. Following the ligation, 50 nM of sample was diluted in Novex® TBE-Urea Sample Buffer (LC6876, ThermoFisher Scientific) to a final volume of 12 μL. Samples were denatured at 70° C. for 3 min, placed on ice for 5 min, 10 μL was loaded onto 15% Novex® TBE-Urea Gel (EC6885BOX, ThermoFisher Scientific) and separated in XCell SureLock™ Mini-Cell Electrophoresis System (ThemoFisher Scientific) using PowerPac Basic Power Supply (Bio-Rad) for ˜90 min at 170V. Gels were stained using 1× SybrGold (S11194, Invitrogen) in 1×TBE running buffer for 15 min followed by imaging in Gel Doc XR System (Bio-Rad). In chimeric probes binding assay, concentrations of template and padlock probes as stated above were incubated with 62 mU/μL PBCV-1 DNA ligase and 0.4 U/μL RNaseinhibitor at room temperature for 10 minutes. Reactions were stopped by adding 1 μL 0.5 M EDTA and 5 μL 100 formamide.

[0264] Results

[0265] Effect of 3′-OH RNA on PBCV-1 DNA Ligase RNA-Dependent Ligation: RNA End Joining for Different RNA Substrates

[0266] We compared the ability of PBCV-1 ligase to circularize padlock probes hybridised to let-7a, where the 5′ end of the probe is DNA, and the 3′ end was either DNA or RNA. While the ability of T4Rnl2 to join chimeric 3′ RNA acceptor strands with 5′ donor strands on RNA is well characterised, this has only been demonstrated for PBCV-1 DNA ligase on DNA templates. We compared the ligation efficiency of chimeric padlock probes versus DNA padlocks probes over reaction time by PAGE separation (FIG. 14). Moreover, we measured the ligation efficiency as total number of rolling circle amplification products (RCP), digitally counted for each padlock/template pair (FIG. 1A).

[0267] PBCV-1 ligase catalysed highly efficient end-joining when 3′ RNA containing PLPs were ligated on miRNA targets (FIG. 1B). For longer non-miRNA targets, ligation efficiency of chimeric and non-chimeric probes was similar (FIG. 2A). Since presence of RNA results in greater nucleic acid duplexes stability, we hypothesised that more stable duplexes would be ligated faster during the initial reaction stage. T4Rnl2 efficiently ligated chimeric padlock probes, while relatively lower activity was seen for DNA padlock probes (FIG. 2B). Our finding that PBCV-1 readily accepts chimeric padlock probes as substrates, motivated us to systematically characterise the ligation fidelity on synthetic targets having a polymorphic position in the centrally located nucleotide (FIG. 3 and table 1). To measure the effect of mismatched chimeric substrates on PBCV-1 and T4Rnl2 ligase end-joining activity, four chimeric padlock probes, differing with a terminal 3′ nucleotide (rA, rU, rG, rC), were hybridised with four different RNA targets each, ligated and amplified with RCA (FIG. 3). PBCV-1 ligase was highly tolerant towards most 3′ RNA mismatches (FIG. 3A, 3B). T4Rnl2 on the other hand, was moderately accurate ligating rC/rG (82%) and rA/rC (64%) but showed poor end-joining fidelity towards other combinations (FIG. 3A, 3C).

[0268] Effect of Various RNA Substitutions on RNA Templated iLock Probe Activation, Ligation Efficiency and Fidelity for PBCV-1 as Well as T4Rnl2

[0269] In our previous study, we have utilized structure-specific 5′ flap cleavage activity of Taq DNA polymerase, used in the invader assay, to activate padlock probe molecules for ligation. We have shown that this iLock probe assay increases ligase-based RNA detection fidelity (Krzywkowski supra). As PBCV-1 ligase was fairly tolerant for majority of the chimeric 3′ mismatches tested, we tested how presence of RNA substitutions in various position of an iLock probe will affect probe activation and ligation, compared to a DNA iLock probe. Multiple iLock probes, targeting let-7a miRNA, were designed (table 2), containing RNA substitutions in various probe positions (FIG. 4A). One chimeric probe (called “3”) had the RNA substitution at the 3′ terminus. In the “3D” probe, the terminal 3′ and the displaced base of the 5′ flap was substituted with RNA. The “3D5” probe had in addition to the substitutions in the “3D” probe, an additional RNA base at the position 3′ to the “D” position. This RNA base would become the 5′-phosphate donor end in a ligation reaction after successful iLock activation. Lastly, we designed a probe with the terminal 3′ and the complete 5′ flap as RNA bases (“3DF”), and a probe where only the 5′ flap was composed of RNA (“DF”) (i.e. lacking the 3′ RNA).

[0270] Compared to a non-chimeric let-7a iLock, iLock-3 greatly increased detection of let-7a miRNA. According to PAGE of ligated iLock probes (FIG. 4C), only a fraction of non-chimeric iLock probes was activated (cleaved) at given conditions and even smaller fraction was ligated (FIG. 4C, lane 4). Virtually all activated iLock-3 probes became ligated as evident by a quantitative gel-shift in the FIG. 4C (lane 5), as well as the total number of RCA products generated with iLock-3 probe (FIG. 4B). When the flap nucleotide displaced by an invading terminal 3′ RNA was substituted with RNA, as in iLock-3D, an additional efficiency increase was observed. The majority of iLock-3D was activated and ligated (FIG. 4C). A similar effect was observed for the iLock-3DF probe, where whole 5′ flap sequence was RNA, while the positive effect was lost in the absence of a terminal 3′ RNA base (FIG. 4B). Interestingly, iLock-3D5 probe, containing 3′-(rN)/5′-(rN) after activation, showed significantly lower performance than an iLock probe with a deoxyribonucleotide at the (5) position. Significantly increased performance of iLock-3D probes was observed for other miRNAs we have tested (miR21) using both PBCV-1 and T4Rnl2 ligase (FIG. 5). Similarly, in a repeated experiment with other iLock-3D probes increased performance was also observed using let-7f as a template, for both PBCV-1 and T4Rnl2 (FIG. 27). To test if accuracy of RNA sensing with chimeric iLock-3D probes is maintained, we targeted the four polymorphic RNA templates (table 2) with four chimeric iLocks-3D probes. Chimeric iLock probes showed excellent fidelity towards matching rC/rG, rA/rU and rU/rA probe pairs (FIG. 6). iLock-3D probes showed no ligation products when templates were omitted. T4Rnl2 displayed full compatibility with the iLock RNA detection assay, readily ligating targets-matching 3′-OH(rN)/5′-p(N) iLock probes (FIG. 6B). rG/rC pair was detected with relatively lower fidelity for both PBCV-1 and T4RN12, showing rG/rU mis-ligation of 5% and 27% respectively (FIG. 6A, 6B). In a further experiment, both PBCV-1 and T4Rnl2 displayed full compatibility with the iLock RNA detection assay, readily ligating targets matching 3′-OH(rN)/5′-p(n) iLock probes (FIGS. 28A-B, FIG. 29, A-B). rG/rC pair was detected with relatively lower fidelity for both enzymes, showing rG/rU mis-ligation of 21% and 11%, respectively. Thus, as can be seen from FIG. 28A-B, chimeric ilocks have better performance than DNA ilocks.

[0271] Multiplexed Detection of Let-7 Isoforms Using Chimeric iLock Probes

[0272] High multiplexing capacity is one of the most advantageous feature of padlock probes. Differentiation of amplified products, originated from different padlock probes, is typically achieved by using unique, probe-specific decorator oligonucleotides, labelled with fluorophores with different emission spectra. Alternatively, unique barcode sequence can be embedded in the padlock probe backbone that can be decoded using next-generation sequencing-by-ligation chemistry. To assess compatibility of chimeric iLock probes with sequencing-by-ligation readout, we have redesigned four let-7 family iLock-3D probes as described in methods section (table 3). To evaluate if barcoded iLock-3D probes could be utilized in multiplexed miRNA profiling, we have combined let-7f, let-7e, let-7d miRNAs in four different stoichiometric ratios. Ideally, ratios would be accurately reflected in miRNAs-specific sequencing reads. The iLock probes were applied in multiplex and the amplified products were fixed onto a glass surface. The barcodes of the RCPs were decoded using sequencing-by-ligation chemistry. Since only three iLock probes were used in this experiment, it was enough to sequence the first barcode position to decode which miRNA was detected. The iLock probes showed similar relative efficiency on the miRNA pool (FIG. 7). In samples where the concentration of one miRNA was increased, the signal increased for the corresponding iLock probe, while the signal for the other targets remained stable (FIG. 7C).

Example 2—Ligation of DNA Padlock Probes, and Chimeric Probes Comprising 1 or 2 Ribonucleotides at their 3′ End

[0273] Materials and Methods

[0274] The ligation reactions were performed with 1 nM final concentration of DNA padlock probe or chimeric padlock probe with either 1 or 2 terminal 3′ ribonucleotide bases, and 2 nM final concentration of synthetic KRAS RNA template or KRAS DNA template. Oligonucleotide sequences are shown in Table 6. Reactions were incubated in ligation buffer containing 1 U/μL RNAseinhibitor, 0.2 mg/mL BSA and 1× SplintR buffer or T4RNA ligase II buffer and 0.25 U/μL (low conc) or 1.25 U/μL (high conc) SplintR ligase, or 0.2 U/μL (low conc) or 1 U/μL (high conc) T4 RNA ligase II, respectively, in final volume of 10 μL for 30 min at 37 C. After that the circles were amplified with Rolling circle amplification in RCA reaction buffer, as described above, with a final circle concentration of 100 pM. Finally, the RCA products were labelled with Cy3-labelled detection probes in final concentration of 10 pM. Labelled RCA products were digitally counted.

[0275] Results

[0276] Both chimeric padlock probes, with 1 or 2 terminal 3′ ribonucleotide bases generate more countable RCA products than pure DNA padlock probes, both for SplintR ligase and T4 RNA ligase II, and both on RNA templates and on DNA templates (FIG. 8). An increased ligase concentration did not have any effect on chimeric padlock probes, but a slightly negative effect on DNA padlock probes. There was no difference in RCA product count between chimeric padlock probes with 1 or 2 terminal 3′ ribonucleotide-bases.

[0277] On RNA templates the activity of Splint R ligase and T4 RNA ligase II are similar (FIG. 8A). On DNA templates the difference between DNA padlock probes and chimeric padlock probes is similarly high for SplintR ligase as on RNA templates, while a very strong increase in RCP counts was recorded for ligating chimeric padlock probes on DNA templates with T4 RNA ligase II (FIG. 8B). T4 RNA ligase II does not accept 3′ DNA and 5′ DNA ends when templated by DNA, but readily accepts probes with 3′ RNA ends when templated by DNA.

[0278] In conclusion, the use of chimeric probes makes ligation reactions with both SplintR ligase and T4 RNA ligase II on RNA templates more efficient than conventional DNA probes. Chimeric probes enable usage of T4 RNA ligase II for ligation reactions on DNA templates.

Example 3—In Situ KRAS Point Mutation Detection Using Chimeric Padlock Probes and Chimeric iLock Probes

[0279] Materials and Methods

[0280] ONCO-DG-1 and A-427 cell lines were cultured in RPMI culture medium without L-Glutamine supplemented with 10% FBS 2 mM L-Glutamine and 1× Penicillin-Streptomycin (PEST). A-549 was cultured in DMEM supplemented 10% FBS and 1×PEST. When confluent, all cell lines were seeded on Superfrost Plus slides and allowed to attach for 12 h. The cells were then fixed in 3% paraformaldehyde in DEPC-treated PBS (DEPC-PBS) for 15 min at room temperature. After fixation, slides were washed twice in DEPC-PBS and dehydrated through an ethanol series of 70%, 85% and 100% for 4 min each. Secure seal chambers were mounted on the slides, the cells were hydrated by a brief wash with PBS-T (DEPC-PBS with 0.05% Tween20 followed by a permeabilization with 0.1 HCl in H.sub.2O for 1 min at room temperature. Cells were washed twice in DEPC-PBS-T and then DNA or chimeric probes in final concentration of 50 nM were added in hybridization buffer containing 2×SSC, 20& Formamide and 0.4 U/μL RNAseinhibitor. Oligonucleotide sequences are shown in Table 6. Probes were hybridized for 60 min at 37° C. Then the probe hybridization mixture was removed and the cells were washed in pre-warmed (37° C.) wash buffer containing 2×SSC and 25% Formamide for 15 min at 37° C. and once in pre-warmed (37° C.) wash buffer containing 2×SSC and 20% Formamide for 15 min at 37° C. Cells were washed once in PBS-T. Then ligation reaction mix was added to the DNA/chimeric padlock probe experiments (FIG. 9) and invader reaction mixture was added to the DNA/chimeric iLock experiment (FIG. 10). Ligation reaction mixture contained SplintR ligase buffer, 0.2 mg/mL BSA, 0.8 U/μL RNAseinhibitor and 0.25 U/μL SplintR ligase. The ligation reaction was incubated for 60 min at 37° C. The invader reaction mixture contained the same as the ligation reaction and additional 0.1 U/μL Taq DNA polymerase. The invader reaction mixture was incubated for 60 min at 37° C. Subsequently, all experimental reactions were washed with PBS-T twice. 100 nM RCA primer was hybridized to the circles in situ in 2×SSC and 20% Formamide hybridization buffer for 30 min at room temperature. The cells were washed twice in PBS-T. Next, RCA reaction mixture was added containing 1× phi29 reaction buffer, 0.25 mM dNTP, 0.2 mg/mL BSA, 1 U/μL phi29 polymerase and 5% Glycerol and incubated at 37 C for 3 hours. Subsequently, cells were washed in PBS-T twice and detection probes were hybridized to the RCA products in situ (Cy3 labelled probes to KRAS wild type probes, Cy5 labelled probes to KRAS mutant probes) in 2×SSC and 20% Formamide hybridization buffer for 30 min at room temperature. Cells were washed in PBS-T three times, the nuclei stained with DAPI, washed three times again and then mounted in Slowfade mounting medium. Cells were imaged in fluorescent microscope with 20× objective and RCA products quantified using Cell profiler software.

[0281] Results

[0282] Chimeric padlock probes resulted in a higher in situ RNA detection efficiency on both A549 and OncoDG1 cell lines compared to DNA padlock probes (FIG. 9B). Moreover, the ratio of specific/unspecific RCPs per cell in both A549 and in OncoDG1 is increased for chimeric padlock probes over DNA padlock probes (more mutant RCPs than wild type RCPs are detected in A549 cells (carrying a KRAS codon 12 point mutation) and more wild type RCPs than mutant RCPs are detected in OncoDG1 cells (KRAS wild type), making it possible to more accurately detect point mutations with chimeric probes directly on RNA in situ.

[0283] In order to further increase the specificity for point mutations, we applied chimeric iLock probes in situ and found specific detection of KRAS wild type mRNA in OncoDG1 cells and detected the KRAS codon 12 point mutation in A549 cells (FIG. 10).

[0284] In summary, chimeric probes strongly increase the in situ RNA detection efficiency, making in situ RNA analysis more sensitive, cost- and time-efficient than classic cDNA approaches. Moreover, compared to DNA padlock probes chimeric padlock probes, and especially chimeric iLock probes show a higher specificity, making RNA analysis in situ more powerful and accurate.

Example 4—Gap Fill iLock Probes

[0285] Materials and Methods

[0286] In-Solution Gap-Fill iLock Reaction:

[0287] The ligation reactions were performed with 10 nM final concentration of Gapfill ILock probe and 30 nM final concentration of synthetic KRAS RNA template (or no template in the negative control). Oligonucleotide sequences are shown in Table 7. Reactions were incubated in reaction buffer containing 1 U/μL RNAseinhibitor, 1 U/μL reverse transcriptase, 25 μM dNTP (or no dNTP in the gapfill negative control), 0.2 mg/mL BSA, 1× SplintR ligase buffer, 0.1 U/μL Taq DNA polymerase and 0.25 U/μL SplintR ligase in final volume of 10 μL for 60 min at 37° C. After that the circles were diluted 10× in PBS-T (to a theoretical concentration of 1 nM) and then amplified with Rolling circle amplification in RCA reaction buffer, as described above, with a final circle concentration of 100 pM. Finally, the RCA products were labelled with Cy3-labelled detection probes in final concentration of 10 pM and digitally counted.

[0288] In Situ Gapfill iLock Reaction

[0289] OncoDG1 cells were prepared and treated, as described in Example 3. Gapfill iLock probes were hybridized to the KRAS RNA in situ in 50 nM concentration with same conditions as in Example 3 After washing, gapfill polymerization invader mixture was added to the cells containing 1 U/μL RNAseinhibitor, 10 U/μL reverse transcriptase, 25 μM dNTP (or no dNTP in the gapfill negative control), 0.2 mg/mL BSA, 1× SplintR ligase buffer, 0.1 U/μL Taq DNA polymerase and 0.25 U/μL SplintR ligase and incubated on the cells for 60 min at 37° C. The cells were washed twice in PBS-T. Next, RCA reaction mixture was added containing 1× phi29 reaction buffer, 0.25 mM dNTP, 0.2 mg/mL BSA, 1 U/μL phi29 polymerase and 5% Glycerol and incubated at 37° C. for 3 hours. Subsequently, cells were washed in PBS-T twice and Cy3 labelled detection probes were hybridized to the RCA products as described above. Cells were washed in PBS-T three times, the nuclei stained with DAPI, washed three times again and then mounted in Slowfade mounting medium. Cells and RCA products were imaged in fluorescent microscope with 20× objective and RCA products quantified using Cell profiler software.

[0290] Results

[0291] RCA products from in-solution gap-fill polymerization-invader reactions were quantified and plotted in in FIG. 11A. When RNA template was present, RCA products were counted, indicating that the reaction worked sufficient. A significantly lower number of RCPs were counted when no template was present (template negative) indicating that gap-fill iLock probes could not be extended, and, hence, the 5′ flap could not be removed and could not be ligated to the 3′ end. In a control reaction, in which RNA template was present but no dNTPs were added, a significantly lower number of RCPs was counted than in the positive reaction (with template and dNTP), indicating that gap-filling through polymerization and triplex formation was limited, significantly reducing the cleavage of the flap, and hence, generating less ligation products (FIG. 11A). The same trend was visible in the in situ reaction (FIGS. 11B-C).

Example 5—Use of Phi29 as a Reverse Transcriptase Enzyme

[0292] Materials and Methods

[0293] Oligonucleotides

[0294] Oligonucleotide sequences are shown in Table 8 and Table 9. Probes were provided as described in Example 1. Ligation reactions were performed on synthetic KRAS mRNA templates. Padlock probes were designed such that upon RNA hybridisation, probe undergoes circularisation forming a nick between terminal arms. For convenient size assessment of rolling circle products (RCP), a reporter sequence was embedded in the sequence linking the probe arms (backbone). Complementary decorators were used for RCP staining by hybridising to the reporter sequence. For real-time RCA assessment, amplified DNA was stained with SybrGold dye.

[0295] Real-time RCA. To assess the effect of RNA bases on reverse-transcription performance by Phi29 polymerase, 20 nM of padlock probes were mixed with 10 nM of RNA template supplemented with 4 U RNase Inhibitor (DNA Gdansk), 3.75 U of PBCV-1 DNA ligase (SplintR, M0375S, NEB) in the respective buffer in a final volume of 15 μL. The reactions were incubated at 37° C. for 30 min. Following the ligation, 2 μL ligation volume (circles) was mixed in 18 μL RCA reaction mix containing 1× Phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 mg/mL BSA (NEB) and 1× SybrGold (S11194, Invitrogen) to a final concentration of 2 nM circles. To ensure simultaneous initiation of RCA across all samples, circles were put in the tube lids and spun down using table-top centrifuge into the pre-disposed master mix. RCA was immediately initiated and SybrGold incorporation monitored using a Mx3005P qPCR System (Agilent Genomics) at 37° C. for 60 min, followed by Phi29 inactivation at 65° C. for 2 min.

[0296] To investigate whether RCA efficiency of RNA-rich circles can be stimulated by addition of reverse transcriptase, 100 U of RNaseH(−) TranscriptME Reverse Transcriptase (DNA Gdansk) were added to the RCA reaction mixture.

[0297] Sequencing of RCA Products Generated from RNA Containing Circles

[0298] Monomerization

[0299] In order to sequence the incorporated bases within the RCA products that correspond to the RNA bases within the circular templates, the RCA products were first monomerized by restriction digestion. First, RCA products from the real-time RCA measurements, as described above, were diluted in PBS-Tween 0.05% to a concentration of 100 pM. Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1× Phi29 DNA polymerase buffer, 2 mg/mL BSA, 100 nM restriction oligonucleotide (AluI KRAS RO—Table 9), 120 mU/μL AluI (NEB) and 10 pM final concentration of RCA products during 10 min incubation at 37° C. and subsequent heat inactivation at 65° C. for 2 min. After complete digestion of the 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 hour RCA of an 80 base circle yields ˜1000× amplification). The RCA monomers were diluted to 100 pM in PBS-Tween 0.05%.

[0300] Sequencing Library Preparation of RCA Monomers

[0301] The RCA monomers were first tagged with Illumina adapter sequences during a PCR reaction, containing 1× Taq DNA polymerase buffer (NEB), 1.5 mM MgCl.sub.2 (NEB), 250 μM dNTP, 1× SybrGold, 25 mU/μL Taq DNA polymerase (NEB), 0.5 μM forward primer PE1 (table 9), 0.5 μM reverse primer PE2 (table 9) and final concentration of 10 pM RCA monomers. The PCR reaction was started with 5 min denaturation at 95° C. and cycled between 95° C. for 15 sec, 55° C. for 30 sec and 70° C. for 20 sec for 20 cycles. The reaction was monitored in the qPCR instrument and the reaction was stopped before the amplification reached saturation. After the first PCR step (Extension step), 1 μl of the PCR products were spiked into index PCR mixture containing 1× Phusion HF Buffer (Thermo Scientific), 0.2 mM d(A,T,G,C)TP (Thermo Scientific), 1% DMSO, 250 nM index PCR primers (table 11), each sample was labelled with unique combination of 1 of 7 different forward and 1 of 3 different reverse index primers) and programmed for an initial 2 min at 95° C., and 2 cycles of 95° C. for 15 sec, 60° C. for 1 min and 72° C. for 1 min, and an extra cycle of 72° C. for 3 min. The indexed PCR products were diluted 200 times into PCR mixture containing 1× Phusion HF Buffer (Thermo Scientific), 0.2 mM d(A,T,G,C)TP (Thermo Scientific), 1% DMSO, 500 nM P5 and P7 primers and programed at 2 min at 95° C., and 15 cycles of 95° C. for 15 sec, 60° C. for 30 sec and 72° C. for 30 sec. The PCR products were pooled and purified using QIAquick PCR Purification Kit and sequenced by NSQ® 500 hi-Output KT v2 (75 CYS) in NextSeq® 550 system (Illumina). The reads containing correct primer sequences at the expected positions were extracted and analysed with WebLogo.

[0302] Morphological Assessment of RCP Size and Intensity

[0303] To measure RCA product size and intensities, the RCA products from the real-time RCA reaction were diluted to a final concentration of 20 pM, labelled with 5 nM final decorator probe concentration in standard hybridization conditions. 10 μL of the fluorescently labelled RCA products were applied to Superfrost glass slides (Thermo Fisher), spread out by a 20×20 mm coverslip (Menzel) and left to bind electrostatically to the positively charged surface during 15 min incubation. Coverslips were removed, slides were briefly washed in PBS, mounted in mounting medium and imaged on a Zeiss Axioplan fluorescent microscope with 20× magnification in the Cy3 channel. Images were exported as original black-white (BW) pictures and processed with Cell Profiler software. Briefly, each image was pre-processed using automated top-hat filtering. Objects were identified using manually adjusted thresholding and separated based on objects intensity. The average fluorescence intensity and object size was recorded, exported as a csv file and processed in R! Studio.

[0304] Results

[0305] Phi29 DNA Polymerase Accepts Chimeric Circles as Rolling Circle Amplification (RCA) Templates

[0306] We have observed that circularized chimeric padlock probes containing both RNA and DNA nucleotides can be used as substrates for RCA, suggesting that Phi29 DNA polymerase possesses a reverse-transcriptase activity. To investigate this activity, we have circularized a variety of RNA/DNA chimeric padlock probes containing 1-7 RNA substitutions in a DNA probe backbone and used the circularized probes as templates during RCA (FIG. 13).

[0307] The RCA reaction was monitored in real-time by SybrGold incorporation. Additionally, the RCA products were digitally counted and their size and intensity (i.e. morphology) assessed.

[0308] We observed that PBCV-1 readily sealed both pure DNA probe and chimeric 3′-(rN)/5′(N) probe nicks and that Phi29 polymerase accepted both pure DNA and RNA-containing circles as templates for RCA (FIG. 13A). The higher number of RCA products from the 3′ RNA probes is most likely due to increased ligation efficiency of chimeric probes, as described in detail in Example 1. When no template is added during the ligation reaction, probes are not ligated and cannot be amplified (FIG. 13A, target -).

[0309] Next, we aimed to investigate RCA efficiency of chimeric DNA/RNA circles without the bias of the ligation reaction. For this purpose we added RNA substitution in the circle backbone that do not participate in the ligation reaction. Consequently, all probes contained the same target-complementary DNA probe arm sequences (contributing to forming the ligation substrate) and were ligated on the same RNA target. We then investigated how an increasing number of RNA substitutions affect the RCA reaction efficiency by counting RCA products (FIG. 13B) and monitoring the amplification reaction in real-time (FIG. 13C). Padlock probes having sequences SEQ ID NOs:119, 120, 124, 131, 110, 111 and 112, being a DNA probe and chimeric probes having 0, 1, 2, 3, 4, 5 and 7 consecutive ribonucleotides, were used.

[0310] We have observed no effect on the RCA efficiency when a single RNA was substituted in the probe (circle) backbone (FIGS. 13B and 13C, FIGS. 32A and 32B). A strong inhibition of RCA (approximately 90%) was observed when circles were substituted with 2 consecutive RNA nucleotides (FIGS. 13B and 13C, FIGS. 32A and 32B). For circles with more than 2 RNA substitutions, no amplification was detected (FIG. 13E, FIG. 32C). When rolling circle products (RCP) were imaged using epi-fluorescence microscopy, the average size and intensity of RCPs was decreased for circles with 2 consecutive RNA substitutes (FIG. 13D). More than two consecutive RNA substitutions (3-7) led to complete RCA inhibition and no RCPs were detected in these samples.

[0311] We have additionally investigated whether RCA of RNA-rich circles can be recovered by supplying a M-MuLV reverse transcriptase during the RCA reaction. However, under the reaction conditions used, RCA activity did not recover in the presence of the reverse transcriptase. In contrast, the amplification rate was significantly decreased by addition of M-MuLV reverse transcriptase (lower panel of curves) in comparison to RCA reaction without additional reverse transcriptase (FIG. 19).

[0312] Phi 29 DNA Polymerase Preferentially Reverse Transcribes RNA Pyrimidines During RCA

[0313] In the previous experiment, DNA bases in the circles backbone were increasingly substituted with RNA bases. Strong amplification inhibition was observed for circles with rGrA and rGrArC substitutions. In order to study if there is some sequence dependency in RCA efficiency of RNA containing circles, we monitored RCA rate in real-time using circles with single rU/rA/rC/rG RNA base, as well as, di- and trinucleotide long homo-nucleotide stretches (Table 9). We observed efficient RCA for all single RNA substitutions (FIG. 18A). For the dinucleotide RNA substitutions, we observed the highest RCA rate for rCrC circles followed by rUrU circles, while rArA and rGrG circles were substantially inhibited. For trinucleotide RNA circles, only rCrCrC circles generated detectable RCA, however, at a rate substantially slower than rCrC-containing circles (FIG. 18A). A number of the ribonucleotide probes SEQ ID NOs: 108-112, 124-127, 131-134, 137 and 139 contained further hetero-nucleotide ribonucleotide stretches. Experiments were repeated using probes SEQ ID NOs:267-281 (Table 13) which did not have the additional ribonucleotide stretches and similar results were observed (FIGS. 24A-D and FIG. 25).

[0314] To investigate whether RCA can be recovered for longer mixed RNA/DNA stretches, we interspaced 1 and 2 DNA bases in stretches of 3 and 6 RNA bases, respectively (FIG. 18 B). Circles containing rGArCGrU sequence in the backbone were amplified, while no RCA was detected for circles with 6 interspaced RNA substitutions, or for circles with 5 and 7 consecutive RNA bases (FIG. 18B).

[0315] Manganese Ions Increase RNA-Dependent RCA Activity of Phi29 DNA Polymerase

[0316] As certain DNA-dependent DNA polymerases are able to reverse-transcribe RNA in the presence of Mn.sup.2+, we compared phi29 DNA polymerase RCA rates with Mg.sup.2+ and Mn.sup.2+. Using Mn.sup.2+ as a cofactor, phi29 DNA polymerase in addition to rC also efficiently amplified single, dinucleotide and trinucleotide rU and rA stretches (FIGS. 24A-D). Interestingly, amplification rates of rCrC-, rUrU and rArA-circles were higher when compared to single RNA substituted circles, which was also true for rCrC with Mg.sup.2+ as cofactor (FIGS. 24A-C). To investigate whether RCA can be recovered if multiple RNA bases are mixed with DNA-bases, multiple chimeric constructs were amplified with Mg.sup.2+ and Mn.sup.2+ (FIG. 25). According to our observations, phi29 DNA polymerase was able to engage in efficient Mn.sup.2+-dependent RCA when RNA bases were interspaced with DNA (FIG. 25), Table 12). Interestingly, circular chimeric substrate with as many as 8 RNA bases was well amplified when substitutions were organised in a uniformly dispersed pattern (FIG. 25).

[0317] Sequencing of Rolling Circle Products Demonstrates Ability of Phi29 DNA Polymerase to Reverse Transcribes RNA

[0318] Since the Phi29 polymerase amplification rate was inversely proportional to the number of RNA bases in the substrate, we considered that the enzyme may ignore RNA positions during RCA, introducing single, or double nucleotide deletions in amplified product. To validate this hypothesis, circles containing single and double RNA substitutions (rAr/Ur/G/rC/rArA/rUrU/rGrG/rCrC, Table 9) were amplified and RCA rate was monitored in real-time as described earlier. Following amplification, sequencing libraries were prepared from the different amplification products, and then sequenced using Illumina NextSeq® 550 system. Full length sequencing reads were extracted from the dataset, sequencing reads were aligned and base frequency was calculated for each position in the padlock probe backbone (FIG. 20).

[0319] According to our observations, Phi29 DNA polymerase incorporated the expected DNA nucleotides in the amplified RCP where template sequence was RNA. For circles with no RNA substitutions, >99% of sequenced monomers showed correctly incorporated base at R1 padlock probe region (here called accuracy) highlighted in the FIG. 20 (99.68%, RT accuracy for position R1 for DNA padlock probe). When R1 position was substituted with rA, rC, rG or rU, the RT accuracy was 99.88%, 99.70%, 96.07%, 99.88% respectively, While the rA, rC, and rU was copied with better accuracy than sequencing, or at least not worse than for dG in the investigated position, rG stands out with higher replication error. Interestingly, this higher incorporation error was observed not only for the R1 position, but for all following cytosine bases in the padlock probe backbone. When both R1 and R2 positions were substituted with rArA, rCrC, rGrG and rUrU, RT accuracy for the R1/R2 site was 99.81/99.59%, 99.82/99.86%, 93.01/89.7% and 99.93/99.94% respectively. Similarly to circles with a single rG substitution in R1, all dinucleotide RNA substrates demonstrated higher error rate for non-RNA cytosines across probe backbone sequence.

[0320] In a further experiment, when R1 position was substituted with rA, rC, rG or rU, the average error rate was 0.111%, 0.153%, 2.259% and 0.084% respectively FIG. 30, FIG. 31, FIG. 33). While the rA, rC, and rU was copied with the same accuracy as DNA (as measured by sequencing), rG stands out with higher replication error. Interestingly, this higher incorporation error was observed not only for the R1 position, but for all guanosine bases in the padlock probe backbone (visible as high error rate peaks in FIG. 31 and FIG. 33) and higher thymine frequencies for the rG padlock probe logo graphs in FIG. 30. When both R1 and R2 positions were substituted with rArA, rCrC, rGrG and rUrU, the error rate for the R1/R2 site was 0.269/0.561%, 0.107/0.109%, 2.827/2.231% and 0.144/0.220% respectively. Similarly to circles with a single rG substitution in R1, all dinucleotide RNA substrates demonstrated higher error rate for non-RNA guanosines across probe backbone sequence (FIG. 3C).

[0321] We demonstrate limited reverse transcription activity of Phi29 DNA polymerase. We show that single RNA substitutes in circular templates have no impact on RCA efficiency. We have found, however, that amplification was suppressed when more consecutive RNA bases were substituted in the circular template sequence. In order to characterize this novel activity of Phi29 polymerase, we amplified circular templates containing either one, two, or three consecutive RNA bases rA, rG, rC or rU with Phi29 Polymerase and monitored the RCA rate in real time. Moreover, we tested various combinations of different RNA bases and interspacing RNA bases with DNA bases. Our data demonstrate a preference for circular substrates containing pyrimidine RNA bases, since circles with 3 consecutive pyrimidine bases could still be amplified, but not circles with 3 consecutive purine bases. Interestingly, interspacing circles with 3 RNA substitutions with DNA bases led to a partial recovery of the RCA efficiency, indicating that RCA of RNA containing circles is restricted to single RNA base substitutions or very short stretches of consecutive RNA bases. The attempt to increase RCA efficiency of circles containing longer stretches of RNA bases by addition of reverse transcriptase failed. Instead, RCA was suppressed in the presence of dedicated reverse transcriptase, potentially due to blocking of circular substrates for Phi29 DNA polymerase binding.

[0322] Our data clearly illustrates that the mechanism, by which the polymerase copies RNA containing circles, is reverse transcription, as we found the matching DNA base incorporated into the RCA products with high frequency (>99% for rA, rU and rC, ˜96% for rG). The overall incorporation accuracy on RNA substitutes was not different from the accuracy on pure DNA substrates.

Example 6—In Situ Detection of mRNA Using Chimeric Probes

[0323] Materials and Methods

[0324] BjHtert and MEF cells were cultured in growth medium consisting of Dulbecco's modified Eagle medium (DMEM; Invitrogen), 10% fetal bovine serum (Sigma), and 1% penicillin-streptomycin mix (PEST; Gibco). Both cell lines were grown in a humidified cell incubator at 37° C. in the presence of 5% CO2. Prior the experiments, cells were dislodged from the culture flask using trypsin-EDTA 0.25% solution (T4049 Sigma) and grown in 150-mm cell culture dishes with 5 submerged microscope slides over-night. Slides with the attached cells were washed twice with PBS, fixed in freshly prepared diethyl pyrocarbonate (DEPC)-treated PBS containing 3.4% formaldehyde for 15 min on ice. Thereafter, slides were washed twice with DEPC-PBS, dehydrated in an ethanol gradient (70%, 85%, and 99%; 3 min each), air dried, and stored at 80° C. At the experiment day, cells were thawed, dried and, for each experimental condition tested, isolated by covering with an 8 mm diameter and 50 ul volume Secure Seal chamber (Invitrogen). Cells were rehydrated with DEPC-TBS buffer. Each incubation step was followed by two DEPC-PBS-T washes ((DEPC)-treated PBS containing 0.05% Tween 20 as a surfactant agent). All incubations were performed in a humid chamber to avoid evaporation of the reaction mixture.

[0325] Probes for both ACTB transcripts (Table 4) were pooled and pre-hybridised (pool 1: non-chimeric PLPs, pool 2: chimeric PLPs, pool 3: iLocks, pool 4: chimeric iLocks) in final concentration 0.1 μM in hybridisation buffer (475 mM Tris-HCl at pH 8; 0.95 mM EDTA, 760 mM NaCl are shown in Table 4. 0.8 U/μL RNase Inhibitor (DNA Gdansk) in 50 uL reaction volume at 37° C. for 2 hours. Unhybridised probes were removed by stringent washing using pre-heated (37° C.) TBS-Tween buffer, twice. The ligation reaction was performed by adding 0.5 U/μL SplintR ligase (NEB), 1× SplintR buffer, 0.8 U/μL RNase Inhibitor in DEPC-ddH2O. iLock ligation and activation (for iLock probes) was conducted simultaneously by adding Taq DNA polymerase in final concentration 0.1 U/μL. Slides were incubated 2 hours at 37° C. and washed twice with DEPC-PBS-T.

[0326] Rolling circle amplification reaction was conducted by adding 1 U/μL phi29 DNA polymerase (Monserate), 1× phi29 DNA polymerase buffer, 0.25 mM dNTPs (Thermo Scientific), 0.2 μg/μL BSA (NEB), 5% glycerol and DEPC-ddH2O in 50 μL final reaction volume for 6 hours at 37° C. and washed twice with DEPC-PBS-T.

[0327] Finally, decorator oligonucleotides were hybridised to RCA products at 0.1 μM final concentration in hybridisation buffer (2×SSC, 20% formamide, ddH2O) with Hoechst 33342 (Thermo Scientific) in DEPC-PBS at room temperature for 30 minutes. Cells were washed twice with DEPC-PBS-T, dehydrate by passing through an ethanol series (70, 85, and 99.5% ethanol, each for 3 min) and coverslip were mounted with Slow-Fade medium (Thermo Scientific). Signals in cells were quantified using CellProfiler software and analysed in R!.

[0328] Results

[0329] Though probes were pooled together, only the expected signal was observed in cells (FIG. 21A). Moreover, chimeric padlock probes worked more efficiently (generated more detectable RCA products) when compared to non-chimeric padlock probes. iLock probes generated significantly less signal when compared to conventional padlock probes, indicating that further optimisation of the protocol is required to ensure efficient probe activation and RNA detection in situ. Analysis of the data however, revealed that the expected signal was also observed for chimeric and non-chimeric iLock probes, and signal was also higher for chimeric iLock probes (FIG. 21B).

Example 7—Detection of miR21 on a Solid Support Using DNA PLPs, Chimeric PLPs, and DNA and Chimeric iLock Probes

[0330] In this example, miR21 is immobilised on the slide surface and detected in situ. miR21 is prepared with a 5′ biotin moiety separated from the target sequence with 16×rU linker. miR21 is detected with conventional and chimeric padlock probes as well as non- and chimeric-iLock probes. Target and probe sequences are shown in Table 5.

[0331] Materials and Methods

[0332] 8 mm diameter and 50 ul volume Secure Seal chambers (Invitrogen) were put on neutravidin coated microscope slides (PolyAn). In total, six Secure seal silicone chambers were used. miR21 target (miR21_BIO) was diluted to 50 nM final concentration in 1× labelling solution (2×SSC, 20% formamide) incubated at room temperature, 1 hour on gentle shaking. In one instance, miR21 target was intentionally omitted (negative control). After miR21 was immobilised, chambers were washed with PBS-Tween 20 (0.05%) 3×. Chamber where no ligation or activation was taking place (coating control) was kept in PBS until the end of the experiment.

[0333] Padlock probes, iLock probes and “coating control” probes (antimiR21_FAM) were hybridised to immobilised targets at final concentration 10 pM (padlock and iLock probes) or 50 nM (for antimiR21_FAM probe). Probes were hybridised in the hybridisation buffer (475 mM Tris-HCl at pH 8; 0.95 mM EDTA, 760 mM NaCl at 45° C. for 15 minutes and at room temperature for 3 hours on gentle shaking. Chambers were then washed with PBS-Tween 20 (0.05%) 2×.

[0334] The ligation reaction was performed by adding 0.5 U/uL SplintR ligase (NEB), 1× SplintR buffer, in DEPC-ddH2O. iLock ligation and activation (for iLock probes) was conducted simultaneously by adding Taq DNA polymerase in final concentration 0.1 U/uL. Slides were incubated for 1 hour at 37° C. and washed twice with DEPC-PBS-T.

[0335] Rolling circle amplification reaction was conducted by adding 0.5 U/μL phi29 DNA polymerase (Monserate), 1× phi29 DNA polymerase buffer, 0.125 mM dNTPs (Thermo Scientific), 0.2 μg/pL BSA (NEB), 5% glycerol and DEPC-ddH2O in 50 μL final reaction volume for 3 hours at room temperature and washed twice with DEPC-PBS-T.

[0336] Finally, decorator oligonucleotides were hybridised to RCA products at 0.1 μM final concentration in hybridisation buffer (2×SSC, 20% formamide, ddH2O) in DEPC-PBS at room temperature for 1 hour. Cells were washed twice with DEPC-PBS-T, dehydrated in 99% ethanol for 3 min and coverslip were mounted with Slow-Fade medium (Thermo Scientific). Signals in cells were quantified using CellProfiler software.

[0337] Results

[0338] Our data demonstrates efficient immobilisation of biotinylated miRNA target on neutravidin coated microscope slide as no fluorescence was detected from labelled, complementary probe when miR21 was not immobilised. Results of detection are shown in FIG. 22B. Traditional padlock probes generated ˜7800 RCA products (RCPs)/field of view (fov) while ˜36 000 RCPs/fov were quantified when chimeric padlock probes were used. Concordant with the example where ACTB mRNAs were detected in BjhTERT and MEF cultured cells, iLock probes generated less signal in comparison to padlock probes. Chimeric iLock probes generated ˜3 000 RCP/fov while non-modified iLock probes only ˜195 RCPs/fov.

Example 8—In Situ Multiplexed Gene Expression Profiling and Cell Type Analysis Using Chimeric Padlock Probes and In Situ Sequencing in Mouse Brain Tissue Sections

[0339] Materials and Methods

[0340] A P30 mouse brain was, right after surgical removal and without any fixation, imbedded into OCT medium and directly frozen on dry ice, and thereafter stored at −80° C. until usage. 10 μm sections were then cut with a cryostat and sections collected on Superfrost glass slides. Sections were then shortly fixated in 3.7% PFA in DEPC treated PBS for 5 min at room temperature. After that the sections were washed once in DEPC-PBS Tween 0.05% and permeabilized with 0.1 M HCl for 5 min at room temperature. After the permeabilization, slides were washed twice in DEPC-PBS and dehydrated through an ethanol series of 70%, 85% and 100% for 2 min each. Secure seal chamber was mounted on the slide covering the tissue section, and the tissue was hydrated by a brief wash with PBS-T (DEPC-PBS with 0.05% Tween). To target mRNAs with chimeric padlock probes (PLPs), the section was, after the brief rehydration wash, immersed into chimeric PLP hybridization mixture containing 2×SSC buffer, 20% Formamide, 0.05 M KCl, 0.2 mg/mL BSA, 1 U/μL RNAse inhibitor and 50 nM chimeric PLPs. The hybridization was performed at 45° C. over-night. After that the section was washed 2× in pre-warmed buffer (2×SSC, 20% Formamide) at 37° C. for 15 min. Finally, the section was washed 2× in PBS-T. Then ligation reaction mix was added to the section, containing 1× SplintR ligase buffer, 0.2 mg/mL BSA, 0.8 U/μL RNAse inhibitor and 0.25 U/μL SplintR ligase. The ligation reaction was incubated for 60 min at 37° C. The sections were washed 2× in PBS-T. Next, the section was immersed in rolling circle amplification mixture, containing 1× phi29 polymerase buffer, 0.25 mM dNTPs, 0.2 mg/mL BSA, 1 U/μL phi29 polymerase, 5% Glycerol and 50 nM RCA primer. RCA was performed for 3 h at 37° C. Subsequently, section was washed in PBS-T twice and detection probes (serving as anchor probes in the in situ sequencing reaction) were hybridized to the RCA products in situ in 2×SSC and 20% Formamide hybridization buffer for 30 min at room temperature.

[0341] For in situ sequencing, as previously described in Ke et al. (2013, Nature methods), the section was immersed into sequencing by ligation mixture, containing 1×T4 ligation buffer, 1 mM ATP, 0.2 mg/mL BSA, 0.1 U/μL T4 DNA ligase and 100 nM each of sequencing library base 1 (for sequencing the first barcode position, sequencing library base 2 for sequencing the 2.sup.nd barcode position, etc.). The sequencing reaction was incubated for 1 h at room temperature. The section was then washed 3× in PBS-T and the nuclei stained with DAPI, washed three times again, a short ethanol series was performed as described above, and then the tissue was mounted in Slowfade mounting medium. The tissue section was then imaged in fluorescent microscope with 20× objective. To sequence the 2.sup.nd base the sections were first washed in ethanol to remove mounting medium, then the sections were washed 2× in 100% formamide to strip off the anchor and ligated sequencing probes. The sections were washed 3× in PBS-T and then the sequencing by ligation mix for the second base (same composition as above) was added to the sections and the procedure was repeated for the 3.sup.rd and 4.sup.th position. Images of the sequencing reactions were then processed through Cell profiler software and Matlab scripts, as described previously in Ke et al (Nat methods 2013).

[0342] Results

[0343] Multiplexed in situ gene expression profiling using cDNA synthesis and subsequent targeting of the cDNA by DNA padlock probes (PLPs) is usually limited to high expressed genes, due to the low efficiency of cDNA synthesis. Targeting directly the RNA with PLPs has until now been difficult because of the low probe ligation efficiency of enzymes on RNA and insufficient specificity resulting in false positive signals. In this experiment, we show very efficient ligation of chimeric PLPs on RNA (FIG. 23). We applied chimeric PLPs on mouse brain tissue sections targeting 18 different genes with 5 probes for each gene (90 probes in total) (Table 10). The probes were barcoded with sequencing barcodes that could later be decoded by in situ sequencing. The probes were first hybridized to the RNA and then after a wash, probes were ligated using SplintR ligase. The use of T4RNA ligase 2 may further increase the specificity, as we have shown increased specificity and efficiency with T4 RNA ligase 2 (see previous examples). The ligated probes were amplified with RCA and the barcodes in the RCA products sequenced with sequencing by ligation chemistry, as described previously in Ke et al (Nat methods 2013). The overall expression pattern that was received with the direct RNA approach using chimeric PLPs was very comparable to that received by the traditional cDNA targeting approach (data not shown). For simplicity, general stain of all RCA products is presented in this example. Besides the advantage of high sensitivity for the chimeric PLP direct RNA approach, the assay costs are lower, as the cDNA synthesis step is associated to high costs for the reverse transcriptase, and the assay can be performed faster, since the cDNA synthesis step is omitted. Overall, chimeric probes show a promising potential for highly multiplexed RNA analysis in tissue sections combined with in situ sequencing read-out.

TABLE-US-00001 TABLE 1 SEQ ID 5′ NO: Name modification Sequence (5′-3′)  1 hsa_KRAS rArArCrUrUrGrUrGrGrUrArGrUrUrGrGrArGrCrUrGrGrUrGrGrCrGrUrArGr GrCrArArGrArGrUrGrCrC  2 KRASwt_PLP Phos AGCTCCAACTACCAC(10A)AGTAGCCGTGACTATCGACT(10A)CTTGCCTACGCC ACC  3 hsa_let-7e rUrGrArGrGrUrArGrGrArGrGrUrUrGrUrArUrArGrUrU  4 hsa_let-7d rArGrArGrGrUrArGrUrArGrGrUrUrGrCrArUrArGrUrU  5 let7-a_PLP_1 Phos CTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAAC  6 let7-f_PLP_1 Phos CTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAAT  7 let7-e_PLP_1 Phos CTCCTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAAC  8 let7-d_PLP_1 Phos CTACTACCTCT(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATGCAAC  9 let7-a_PLP_RNA_1 Phos CTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAArC 10 let7-f_PLP_RNA_1 Phos CTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAArU 11 let7-e_PLP_RNA_1 Phos CTCCTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATACAArC 12 let7-d_PLP_RNA_1 Phos CTACTACCTCT(10A)CCTCAATGCACATGTTTGGCTCC(10A)AACTATGCAArC 13 benchm_templ_C rUrCrUrCrGrCrUrGrUrCrArU*rCrCrCrUrArUrArUrCrCrUrCrG 14 benchm_templ_A rUrCrUrCrGrCrUrGrUrCrArU*rArCrCrUrArUrArUrCrCrUrCrG 15 benchm_templ_G rUrCrUrCrGrCrUrGrUrCrArU*rGrCrCrUrArUrArUrCrCrUrCrG 16 benchm_templ_U rUrCrUrCrGrCrUrGrUrCrArU*rUrCrCrUrArUrArUrCrCrUrCrG 17 3′T_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGT 18 3′G_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGG 19 3′A_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGA 20 3′C_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGC 21 3′rT_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGrT 22 3′rG_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGrG 23 3′rA_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGrA 24 3′rC_PLP_2 Phos ATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CGAGGATATAGGrC 25 Decorator probe_1 Cy3 CCTCAATGCACATGTTTGGCTCCa 26 Decorator probe2_2 Cy3 AGTAGCCGTGACTATCGACTa r[N]: RNA oligonucleotide; (10A): linker; a: last four bases of the decorator probe were 2′ O-methyIRNAto prevent oligo hydrolysis by Phi29 polymerase; italics: decorator sequence.

TABLE-US-00002 TABLE 2 SEQ ID 5′ NO: Name modification Sequence (5′-3′) 27 hsa_let-7a rUrGrAGrGrUrArGrUrArGrGrUrUrGrUrArUrArGrUrU 28 let-7a_PLP Phos CTACTACCTCA(7A)CCTCAATGCACATGTTTGGCTCC(7A)AACTATACAAC 29 iLock_1 CGCGTGTCGTTGCCCTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A) AACTATACAAC 30 iLock-3_1 CGCGTGTCGTTGCCCTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A) AACTATACAArC 31 iLock-3D_1 CGCGTGTCGTTGCrCCTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A) AACTATACAArC 32 iLock-3D5_1 CGCGTGTCGTTGCrCrCTACTACCTCA(10A)CCTCAATGCACATGTTTGGCTCC(10A) AACTATACAArC 33 iLock-3DF_1 rCrGrCrGrTrGrTrCrGrTrTrGrCrCCTACTACCTCA(10A)CCTCAATGCACATGTTTGG CTCC(A)AACTATACAArC 34 iLock-DF_1 rCrGrCrGrTrGrTrCrGrTrTrGrCrCCTACTACCTCA(10A)CCTCAATGCACATGTTTGGC TCC(A)AACTATACAAC 35 benchm_templ_C rUrCrUrCrGrCrUrGrUrCrArU*rCrCrCrUrArUrArUrCrCrUrCrG 36 benchm_templ_A rUrCrUrCrGrCrUrGrUrCrArU*rArCrCrUrArUrArUrCrCrUrCrG 37 benchm_templ_G rUrCrUrCrGrCrUrGrUrCrArU*rGrCrCrUrArUrArUrCrCrUrCrG 38 benchm_templ_U rUrCrUrCrGrCrUrGrUrCrArU*rUrCrCrUrArUrArUrCrCrUrCrG 39 3′T_iLock_3 TATATCCCTATATTATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CG AGGATATAGGT 40 3′G_iLock_3 TATATCCCTATATGATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CG AGGATATAGGG 41 3′A_iLock_3 TATATCCCTATATAATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CG AGGATATAGGA 42 3′C_iLock_3 TATATCCCTATATCATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)CG AGGATATAGGC 43 3′U_iLock_ TATATCCCTATATrUATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)C RNA_3 GAGGATATAGGrU 44 3′G_iLock_ TATATCCCTATATrGATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)C RNA_3 GAGGATATAGGrG 45 3′A_iLock_ TATATCCCTATATrAATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)C RNA_3 GAGGATATAGGrA 46 3′C_iLock_ TATATCCCTATATrCATGACAGCGAGA(10A)AGTAGCCGTGACTATCGACT(10A)C RNA_3 GAGGATATAGGrC 47 Decorator Cy3 CCTCAATGCACATGTTTGGCTCCa probe_1 48 Decorator Cy3 TGCGTCTATTTAGTGGAGCCa probe_2 49 Decorator Cy3 AGTAGCCGTGACTATCGACTa probe_3

TABLE-US-00003 TABLE 3 SEQ ID NO: Name Sequence (5′-3′) 50 hsa let-7a rUrGrAGrGrUrArGrUrArGrGrUrUrGrUrArUrArGrUrU 51 hsa let-7f rUrGrArGrGrUrArGrUrArGrArUrUrGrUrArUrArGrUrU 52 hsa let-7e rUrGrArGrGrUrArGrGrArGrGrUrUrGrUrArUrArGrUrU 53 hsa let-7d rArGrArGrGrUrArGrUrArGrGrUrUrGrCrArUrArGrUrU 54 let-7a_seqRNA AAAGATGCGATACrACTACCTCATGCGTCTATTTAGTGGAGCCCGCTATCTTCTTTAACTATACAACCTrA 55 let-7e_seqRNA AAAATGTCGTTGCrCCTCCTACCTCATGCGTCTATTTAGTGGAGCCGCCTATCTTCTTTAACTATACAArC 56 let-7d_seqRNA AAAATGTCGTTGCrCCTACTACCTCTTGCGTCTATTTAGTGGAGCCATCTATCTTCTTTAACTATgCAArC 57 let-7f_seqRNA AAAATGTCGTTGCrUCTACTACCTCATGCGTCTATTTAGTGGAGCCTACTATCTTCTTTAACTATACAArU 58 let-7a_seq AAAGATGCGATACACTACCTCATGCGTCTATTTAGTGGAGCCCGCTATCTTCTTTAACTATACAACCTA 59 let-7e_seq AAAATGTCGTTGCCCTCCTACCTCATGCGTCTATTTAGTGGAGCCGCCTATCTTCTTTAACTATACAAC 60 let-7d_seq AAAATGTCGTTGCCCTACTACCTCTTGCGTCTATTTAGTGGAGCCATCTATCTTCTTTAACTATGCAAC 61 let-7f_seq AAAATGTCGTTGCTCTACTACCTCATGCGTCTATTTAGTGGAGCCTACTATCTTCTTTAACTATACAAT 62 Decorator Cy3-TGCGTCTATTTAGTGGAGCCa probe 63 anchor primer AlexaFluor750-TGCGTCTATTTAGTGGAGCCa 64 seqlibb1T pTNNNNNNNN-FITCb 65 seqlibb1G pGNNNNNNNN-Cy3b 66 seqlibb1A pANNNNNNNN-Cy5b 67 seqlibb1C pCNNNNNNNN-TexasRedb

TABLE-US-00004 TABLE 4 SEQ ID NO: Name Sequence (5′-3′) 68 Mouse ACTB GGCCTGTACACTGACTTGAGACCAATAAAAGTGCACACCTTACCTTACACAAAC 69 Human ACTB TACCTGTACACTGACTTGAGACCAGTTGAATAAAAGTGCACACCTTAAAAATGAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA 70 Mouse PLP TAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTAAAAGTAGCCGTGACTATCGACT GTTTGTGTAAGG 71 Human PLP TAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAAGTCGGAAGTACTACTCTCTTTTTTTTTCATTTT 72 Mouse cPLP TAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTAAAAGTAGCCGTGACTATCGACTGTTTGTGTAAGrG 73 Human cPLP TAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAAGTCGGAAGTACTACTCTCTTTTTTTTTCATTTrU 74 Mouse iLock TAtaTCcctatatGTAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTAAAAGTAGCCGTGACTATCGACTGT TTGTGTAAGG 75 Human iLock TAtaTCcctatatTTAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAAGTCGGAAGTACTACTCTCTTTTTTT TTCATTTT 76 Mouse ciLock TAtaTCcctatatrGTAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTAAAAGTAGCCGTGACTATCGACTG TTTGTGTAAGrG 77 Human ciLock TAtaTCcctatatrUTAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAAGTCGGAAGTACTACTCTCTTTTTT TTTCATTTrU

TABLE-US-00005 TABLE 5 78 miR21_BIO rUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrArGrCrUrUrArUrCrArGrArCrUrGrArUrGrUrUrGrA 79 >miR21B2DO_PLP /5Phos/CTGATAAGCTAGCCGAATCTAAGAGTAGCCGTGACTATCGACTAAAACTACACCA 80 >miR21B2DO_iLock ccgtcgctgcgtTCTGATAAGCTAAAAAAAAGTAGCCGTGACTATCGACTAAAAAAATCAACAT CAGT 81 >miR21 B2DO_rPLP /5Phos/CTGATAAGCTAGCCGAATCTAAGAGTAGCCGTGACTATCGACTAAAACTACACCA TCAACATCAGrU 82 >miR21B2DO_RiLock ccgtcgctgcgtrUCTGATAAGCTAAAAAAAAGTAGCCGTGACTATCGACTAAAAAAATCAACA TCAGrU 83 >B2DO_Cy3 /Cy_3/AGTAGCCGTGACTATCGACT 84 >antimiR21_FAM FAM/TCAACATCAGTCTGATAAGCTA antimiR21 FAM is a “coating control” complementary to miR21, for visualising successful miR21 coating on the slide

TABLE-US-00006 TABLE 6 85 KRAS DNA ACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCT template GGTGGCGTAGGCAAGAGTGCCTTGACGATA 86 wt KRAS PLP AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 87 KRAS RNA rArArCrUrUrGrUrGrGrUrArGrUrUrGrGrArGrCrUrGrGrUrGrGrCrGrUrArGrGrCrArArGr template ArGrUrGrCrC 88 chim wt KRAS PLP AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACrC 89 chim wt KRAS AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACGCGTTCAGTGA PLP_2 TGCCCTTGCCTACGCCArCrC 90 mut KRAS PLP AGCTCCAACTACCACAACCTCAATGCACATGTTTGGCTCCCAGACGTAACGCGTTCAG TGATGCCCTTGCCTACGCCACT 91 chim mut KRAS AGCTCCAACTACCACAACCTCAATGCACATGTTTGGCTCCCAGACGTAACGCGTTCAG PLP TGATGCCCTTGCCTACGCCACrU 92 RCA primer CAT CAC TGA ACG C*G*T 93 mut DO-Cy5 CCTCAATGCACATGTTTGGCTCC 94 wt DO-Cy3 AGTCGATAGTCACGGCTACT 95 KRAS wt invader TAtaTCcctatatrCAGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAAC chim 3-5 GCGTTCAGTGATGCCCTTGCCTACGCCACrC 96 KRAS wt invader TAtaTCcctatatCAGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACG chim 3 CGTTCAGTGATGCCCTTGCCTACGCCACrC 97 KRAS mut invader TAtaTCcctatatrUAGCTCCAACTACCACAACCTCAATGCACATGTTTGGCTCCCAGACGT chim 3-5 AACGCGTTCAGTGATGCCCTTGCCTACGCCACrU 98 KRAS mut invader TAtaTCcctatatTAGCTCCAACTACCACAACCTCAATGCACATGTTTGGCTCCCAGACGTA chim 3 ACGCGTTCAGTGATGCCCTTGCCTACGCCACrU

TABLE-US-00007 TABLE 7  99 KRAS gapfill invader TAtaTCtctatatAGCTCCAACTACCACAAGTAGTCGATAGTCACGGCTACTCAGACG TAACGCGTTCAGTGATGAGTAGGC ACTCTTGCCTAC 100 invader_splint_chim TAtaTCtctatatGCCACrC 101 invader_splint TAtaTCtctatat GCCACC 102 splint GCCACrC 103 splint_chim GCCACC 104 RCA primer CAT CAC TGA ACG C*G*T 105 DO-Cy3 AGTCGATAGTCACGGCTACT

TABLE-US-00008 TABLE 8 SEQ ID 5′ NO Name modification Sequence (5′-3′) 106 Kras_wt_RNA rArArCrUrUrGrUrGrGrUrArGrUrUrGrGrArGrCrUrG rGrUrGrGrCrGrUrArGrGrCrArArGrArGrUrGrCrC 107 R_KRAS wt1_1 Phos AGCTCCAACTACCACAA[1]CArGACGTAACGC GTTCAGTGATGCCCTTGCCTACGCCACC 108 R_KRAS wt1_2 Phos AGCTCCAACTACCACAA[1]CArGrACGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 109 R_KRAS wt1_3 Phos AGCTCCAACTACCACAA[1]CArGrArCGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 110 R_KRAS wt1_4 Phos AGCTCCAACTACCACAA[1]CArGrArCrGTAAC GrCrGTTCAGTGATGCCCTTGCCTACGCCACC 111 R_KRAS wt1_5 Phos AGCTCCAACTACCACAA[1]CArGrArCrGrUAA CGrCrGTTCAGTGATGCCCTTGCCTACGCCACC 112 R_KRAS wt1_7 Phos AGCTCCAACTACCACAA[1]CArGrArCrGrUrA rACGrCrGTTCAGTGATGCCCTTGCCTACGCCACC 113 R_KRAS wt1_6S Phos AGCTCCAACTACCACAA[1]CArGrACGrUrAAC rGrCGTTCAGTGATGCCCTTGCCTACGCCACC 114 R_KRAS wt1_DNA Phos AGCTCCAACTACCAC(10A)[2](10A)CTTGCCT ACGCCACC 115 R_KRAS wt1_RNA Phos AGCTCCAACTACCAC(10A)[2](10A)CTTGCCT ACGCCACrC 116 Decorator probe [1] Cy3 CCTCAATGCACATGTTTGGCTC.sup.a 117 Decorator probe2 [2] Cy3 AGTCGATAGTCACGGCTACT.sup.a r[N]: RNA oligonucleotide; (10A): linker; .sup.a: last four bases of the decorator probe were 2′ O-methylRNA to prevent oligo hydrolysis by Phi29 polymerase; [1/2] decorator binding site and sequence

TABLE-US-00009 TABLE 9 SEQ ID NO: Name Sequence (5′-3′) 118 Kras_wt_RNA rArArCrUrUrGrUrGrGrUrArGrUrUrGrGrArGrCrUrGrGrUrGrGrCrGrUrArGrGrCrArArGrArG rUrGrCrC 119 R_KRAS.0 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACC 120 R_KRAS.1G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACC 121 R_KRAS.1C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACC 122 R_KRAS.1A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArAACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACC 123 R_KRAS.1U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACC 124 R_KRAS.2G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGCGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 125 R_KRAS.2C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCCGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 126 R_KRAS.2A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArACGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 127 R_KRAS.2U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUCGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 128 AluI KRAS RO CGCCACCAGCTCCAACTA 129 PE1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGGTGGCGTAGGCAAGGGC 130 PE2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCAACTACCACAAAGTCG 131 R_KRAS.3G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGrGTTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 132 R_KRAS.3C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCrCGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 133 R_KRAS.3A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArArAGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 134 R_KRAS.3U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUrUCGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 135 R_KRAS.5 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCrGrUAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 136 R_KRAS.6S AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrACGrUrAACrGrCGTTCAGTGAT GCCCTTGCCTACGCCACC 137 R_KRAS.3 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCGTAACrGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 138 R_KRAS.3S AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGArCGrUAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 139 R_KRAS.2 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrACGTAACGrCrGTTCAGTGAT GCCCTTGCCTACGCCACC 140 R_KRAS.7 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCrGrUrArACGCGTTCAGTGAT GCCCTTGCCTACGCCACC r[N]: RNA oligonucleotide, bolded; italic: highlighted region where primer hybridises to the monomer, underlined: extension sequences highlighted that introduce binding sites for index primers; yellow: AluI restriction site, these padlock probes were used in the monomer sequencing experiment.

TABLE-US-00010 TABLE 10 141 Detection probe 5′ Alexa750-TGCGTCTATTTAGTGGAGCC (seq anchor probe) 142 Seq library 1.sup.st base-G 5′ phos-GNNNCTATC-3′ Cy3 143 RCA primer 5′-GGCTCCACTAAATAGACG*C*A-3′ thiophosphate (*) 144 Seq library 1.sup.st base-A 5′ phos-ANNNCTATC-3′ Cy5 145 Seq library 1.sup.st base-C 5′ phos-CNNNCTATC-3′ Texred 146 Seq library 1.sup.st base-T 5′ phos-TNNNCTATC-3′ Alexa488 147 Seq library 2.sup.nd base-G 5′ phos-NGNNCTATC-3′ Cy3 148 Seq library 2.sup.nd base-A 5′ phos-NANNCTATC-3′ Cy5 149 Seq library 2.sup.nd base-C 5′ phos-NCNNCTATC-3′ Texred 150 Seq library 2.sup.nd base-T 5′ phos-NTNNCTATC-3′ Alexa488 151 Seq library 3.sup.rd base-G 5′ phos-NNGNCTATC-3′ Cy3 152 Seq library 3.sup.rd base-A 5′ phos-NNANCTATC-3′ Cy5 153 Seq library 3.sup.rd base-C 5′ phos-NNCNCTATC-3′ Texred 154 Seq library 3.sup.rd base-T 5′ phos-NNTNCTATC-3′ Alexa488 155 Seq library 4.sup.th base-G 5′ phos-NNNGCTATC-3′ Cy3 156 Seq library 4.sup.th base-A 5′ phos-NNNACTATC-3′ Cy5 157 Seq library 4.sup.th base-C 5′ phos-NNNCCTATC-3′ Texred 158 Seq library 4.sup.th base-T 5′ phos-NNNTCTATC-3′ Alexa488 Chimeric probe name RNA directed chimeric probe sequence 159 Calb2_1119_RNA CATCGCAGCGGAGACGACAGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAATGCTATCTTCTTTATACAGCGAAGGAACTCATrG 160 Calb2_1328_RNA CACACACGTCAAGAACACAACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAATGCTATCTTCTTTGAAGCCAAAGAGAAAAGGArA 161 Calb2_164_RNA ACCTTCAATGTACCCATTTCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAATGCTATCTTCTTTAAGAAGTTCTCTAGCTCTTrU 162 Calb2_500_RNA AGGTTCATCATAGGGCCTGTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAATGCTATCTTCTTTTGGGTGTACTCCTGGAGCTrU 163 Calb2_937_RNA GTCAAGAGAGTCAGGACAGCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAATGCTATCTTCTTTGGGGAGGTCTGGGAAGGAGrU 164 Calb2_DO3_CCAA_RNA CTCATACAGATCCTTCAGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCCAACTATCTTCTTTTTCATCTCCTTCTTGTTCTrU 165 Chodl_1164_RNA CGGGCTAGTTTTTGATCTTCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTATCCACAGTGTAGACTGATrU 166 Chodl_1798_RNA AAAGCAAAGAAACAGAACAACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTTCCTAAACTTTATCGAACCrC 167 Chodl_2071_RNA ATTCTATAGGCAACATGTGACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTACTCTGGGGAGCTATTTGCrA 168 Chodl_2252_RNA GTTCTGCTTAGCATCACACTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTTTAATCATTAATATCAGTGrU 169 Chodl_293_RNA TCCTACTCCCTCCTTCCCAGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTTTCCTTGCTTTCCTGCTGGrG 170 Chodl_789_RNA GAACTGGGAGCTGCTTCCATCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTTCATCAGTGTACCAGTTTCrG 171 Chodl_916_RNA TGTTGCACCTGTCGTCATTCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAGTACTATCTTCTTTGCAGATGTAATTGTGCTTCrA 172 Cort_326_RNA CCCGGGGGTACCCCCTCCGACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTGATCTATCTTCTTTCTTTCCTGGCTCTTGGACArG 173 Cort_529_RNA CAAAGCTGACATAAGAAGAACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTGATCTATCTTCTTTTTCTCACAGGGCAGGGGAGrG 174 Htr3a_1014_GGAC_RNA AGTGTGTCTGACACGATGATCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCGGACCTATCTTCTTTTACCGATGGCCGTTGCTGGrC 175 Htr3a_1309_GTAA_RNA ATGGCTGCAGTGGTTTCCCACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCGTAACTATCTTCTTTAAGTCCTGAGGTCCTCCAArC 176 Htr3a_1573_TAAC_RNA CCAAATGGACCAGAGAGTGACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTAACCTATCTTCTTTGTGCCCACTCAAGAATAATrG 177 Htr3a_1750_TATT_RNA AAGTCAGAGAGACAGACTGGCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTATTCTATCTTCTTTGCTTTAAAGCCATGATAGGrG 178 Htr3a_1927_TCGC_RNA GCAAGACAATTTGCTTTTCTCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTCGCCTATCTTCTTTCAGAAGTCTCAGGCATCTArU 179 Htr3a_2045_TGGA_RNA ATTATCCCCTGCTCCCATTGCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTGGACTATCTTCTTTTTAAGATATCATAGCATTTrU 180 Htr3a_21_AAGT_RNA GTCCCAGGCAGACTGCTTTTCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCAAGTCTATCTTCTTTCCACCCGCTGCCAACCTCArU 181 Htr3a_247_CAGC_RNA GTCTGACAGCCTTAGTAGAGCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCCAGCCTATCTTCTTTTTGTAGTTAGCCAGGAGGTrG 182 Htr3a_424_CGGT_RNA AGTCCACTGCAGAAACTCATCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCCGGTCTATCTTCTTTACATTGTCGAAGTCCTCAGrG 183 Htr3a_89_CACT_RNA CTCAGAGCAGCCACTCAGGACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCCACTCTATCTTCTTTCTTCCCAGATGTGGGAGGGrC 184 Htr3a_955_GCTC_RNA AGAGACTCTCTCACCGCTGTCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCGCTCCTATCTTCTTTAGAAGGAGTGTGATCTTGArA 185 Neurod6_1033_RNA TAGAAGGATTCATATGCACTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTACTCAGGGGAGGTACTTTCrA 186 Neurod6_108_RNA CTGCTAGTGACGTCACAGGGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTAGAGCTGGTACCCATGCCArU 187 Neurod6_1524_RNA TGTGATACAGACAAGAGGGACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTAGAGAGAGAGAGAATCACArG 188 Neurod6_168_RNA TCTCATTGATCTCTAAAAAGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTATCTGTGTGTATCTGCACTrA 189 Neurod6_168 8_RNA AGACATTGAAGTATGCTGTGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTCATAACTGTACAACTGAAArU 190 Neurod6_2041_RNA AACAATACAAAACAAGTGCTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTACCTGTACAGAAAAATCCTrG 191 Neurod6_228_RNA TTTTCAGGCTGAGTGTCGCACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTCATTTTGGATCTTCCAAATrC 192 Neurod6_315_RNA GTAGTGTTAACATGGTTCTTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTTACGACAGACTCGTCAAACrG 193 Neurod6_495_RNA TGTCTTCTTCCTCCTCTTCTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTATTCTCATCTTCTTCCTCTrC 194 Neurod6_648_RNA TGTCCAGAGCATCATTGAGGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTGGGGACCACTTTTCGCAAArU 195 Neurod6_714_RNA GTCGTAAAGTTTCTATTTTGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTCCAGATGTAATTTTTGGCCrA 196 Neurod6_971_RNA CCCATGCCCTGGGGGAGTGGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCTTGCCTATCTTCTTTGACTTGGAATTATCAAGAGrU 197 Nov_DO2_ATGC_RNA GGAGAAAGTTCATGACACTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATGCCTATCTTCTTTGAGTCGGTTTGTCTATAArU 198 Pcp4_120_RNA TCAGAAGGCAATGCTCAGGGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCATCCCTATCTTCTTTGCTAGGTCCCACAGAACAGrC 199 Pcp4_181_RNA TCCGGCACTTTGTCTCTCACCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCATCCCTATCTTCTTTTTGTCTTTTCCGTTGGTCGrC 200 Pcp4_305_RNA ACTGAGACTGAATGGCCACACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCATCCCTATCTTCTTTTTTCTTCTGGAATTTTCTGrA 201 Pcp4_386_RNA AACTTGGTGTCTTCAGGTGGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCATCCCTATCTTCTTTTTCTTGATGGATGGTGGTTrG 202 Pcp4_472_RNA TTCAGGTTTGTAGCAGGGTGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCATCCCTATCTTCTTTATGGGTTTCTCTTCATGCArU 203 Pde1a_1081_RNA TTCTCTGTTGAGTCCGTCAGTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTCTATAAGAGGAATGACAATrU 204 Pde1a_120_RNA ACTTTGGTTTTTCTTCAGGCTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTAGCATGGACAATGCTGCGArA 205 Pde1a_1216_RNA TCACACACGGAGCCTTTTGTTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTAGTCTGGGGCATAGCTCCCrA 206 Pde1a_273_RNA CATTTAAGGCAAATACATCATCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTGCTATGCTCCCCGCTTGCTrU 207 Pde1a_334_RNA AGATCATATCTGGTAAAGAGTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTGAATCTTGAAGCGGTTGATrA 208 Pde1a_469_RNA ATATAATGCACAGTTTGAGTTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTTGATACCTGTATGAAGCATrU 209 Pde1a_615_RNA TGTACAGAATAGCAACGTCCTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTCTCAAGCACTGAGCGGTCGrU 210 Pde1a_759_RNA CTGTCGCTAAGACCATTTCATCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTCTGAAAATGCCCTGACATGrU 211 Pde1a_904_RNA CTGTAGTGCAACTTCCAAGTTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTCCATTAGGGCCATGGTCCArU 212 Pde1a_995_RNA CTTCCGATCACAAAGTGGAGTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCATCGCTATCTTCTTTGACTGGGCAACCATTGTTGrA 213 Penk_1282_RNA CAATACTGAGCTTCAAGACTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCGTACCTATCTTCTTTACAACATAGCCATAAGAGArC 214 Penk_286_RNA CATGGGCTGTAGGAGAGAAGTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCGTACCTATCTTCTTTCAAAGCCTCAGGAACCGCGrC 215 Penk_638_RNA GATATAGCTCGTCCATCTTCTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCGTACCTATCTTCTTTTTCTTCTTCTGGCTCCATGrG 216 Penk_83_RNA TGCCTGGGACTATTCTATCTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCGTACCTATCTTCTTTGTTTCCTGCTGTTCTAGTGrA 217 Penk_882_RNA AGTTGGGGGCTTCTTTTGAGTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCGTACCTATCTTCTTTGCTCTTTTGCTTCATCTTCrC 218 Plcxd2_DO3_GAAA_RNA GCACTCCTACACAATGACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCGAAACTATCTTCTTTGAAGATGGTGAGGGTArU 219 Plcxd2_DO3_GGCC_RNA GTGGTAGAAAATGAGAACCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCGGCCCTATCTTCTTTTGCTTGTAGAAGGGACrA 220 Rorb_2282_RNA TCATTCAGAATTGGATTCCACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAAATCTATCTTCTTTATAACCACCAAAGTGAAGTrU 221 Rorb_4479_RNA TCAATTTTCTGCCTTAAGCCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAAATCTATCTTCTTTAAGAAGAAAAAGAAGTTCArU 222 Rorb_536_RNA ATGTGAGGTCATAGATAGGTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAAATCTATCTTCTTTGGTAAACAAGTTGGGTACArG 223 Rorb_6395_RNA GAGAAAGTGTCACAGATTTGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAAATCTATCTTCTTTAGGTACAATTAAGAGAAAGrG 224 Rorb_8435_RNA TAGTTGTTAGGGAGTGCTGCCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCAAATCTATCTTCTTTAAGTAATAGAAAACTCTTTrU 225 Rorb_D03_CCGG_RNA AGCCTTTTAAAGTCATATTTGGTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCCGGCTATCTTCTTAATCGGTCATCATAAAATACrU 226 Rprm_654_RNA CGGTCCGTGATGGTGCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTTGTCTATCTTCTTTGACAGGTTTGCGTTGCrU 227 Sst_432_APlSWPLP_RNA TAACAGGATGTGAATGTCTTCTACGATTTTACCAGTGGCCTGACTATCTTCTTTTAGGACAACAATATTAAAGrC 228 Synpr_1071_RNA CATACTAGAGACTTTAAGCTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATTACTATCTTCTTTAAGGTAATCTATGCACATTrA 229 Synpr_1643_RNA CCTCTCTGGATGCAAAGAAATCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATTACTATCTTCTTTAACTATGGTGTCTAAATCTrG 230 Synpr_260_RNA ATGTACACGACCGTGGCAGCTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATTACTATCTTCTTTGGTACTTGTTCTGGAAGAArA 231 Synpr_591_RNA AAGGTATCTCTGTCCAGAGGTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATTACTATCTTCTTTTGCTTCTCCATGGGGTCTGrA 232 Synpr_DO2_ATTA_RNA CTTAAAAATTCTTCTGCTACTGGTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCATTACTATCTTCTTCATTAATAATTGATTGAAACrU 233 Yjefn3_138_RNA CACTAGCGTGCCCACATAGTCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTTTACTATCTTCTTTGGCCTTGGTCACAGCCACCrG 234 Yjefn3_344_RNA CTTCTCGCACTGCGTGGTCACGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTTTACTATCTTCTTTGACAGGAAGGGGATGTCCArU 235 Yjefn3_686_RNA AAACTTTCGGCGGACGTCATCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGTGGAGCCTTTACTATCTTCTTTTATTTTGGCAGGTGCAGGCrC 236 Bgn_412_RNA GGGCACAGTCTTCAGACCCATCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCCTATCTATCTTCTTTGTGTCAGGTGAGATCTCCTrU 237 Bgn_851_RNA TCAGGGTCTCAGGGAGATCTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCCTATCTATCTTCTTTGTGGTCCAGGTGAAGTTCGrU 238 Bgn_1194_RNA TCCCAGTAGGGCACAGGGTTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCCTATCTATCTTCTTTGGAAGGTGGCAGGCTGCACrU 239 Bgn_1577_RNA AACAATGGCGGTGGCAGTGTTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCCTATCTATCTTCTTTAGGAACACATGCCTGGATGrG 240 Bgn_2309_RNA TCAGGGACCCAGGGGTGAGGTCTACGATTTTACCAGTGGCTGCGTCTATTTAGTGGAGCCCTATCTATCTTCTTTGACCATCACCTCCTACCACrA 241 Apq4_879_RNA CTTAAGGCGACGTTTGAGCTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCATTCTATCTTCTTTGCGGCTTTGCTGAAGGCTTrC 242 Apq4_2186_RNA AATTACACTCACAATGCCGACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCATTCTATCTTCTTTTAATTCACACAAATGGGTArU 243 Apq4_3100_RNA CACTGGAAATGACTGTTAAACTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCATTCTATCTTCTTTTGTACCATACTGAATGCTGrU 244 Apq4_3673_RNA CGGTGTATCTGTCAGTAGCTCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCATTCTATCTTCTTTTTTCCTCTCTGATCTCTGTrG 245 Apq4_4344_RNA ACAGAGGCAGTGTCTCTGTGCTGATTCCTTTGACTCACATTGCGTCTATTTAGTGGAGCCCATTCTATCTTCTTTGCTCTCTGGCTTCAATTGTrC 246 Pdgfra_296_RNA TGGGAGGATAGAGGGTAATATCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCTGTCCTATCTTCTTTACAATCTTCTCATTCTCGTrU 247 Pdgfra_646_RNA GGTATGATGGCAGAGTCATCTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCTGTCCTATCTTCTTTCCGGATCTGTGGTGCGGCArA 248 Pdgfra_832_RNA GTTGCTTTCAAGGCATAAACTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCTGTCCTATCTTCTTTTCTCCAGATTCAGTTCTGArC 249 Pdgfra_1227_RNA GGTAGGCCTGCACCTCCACCTCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCTGTCCTATCTTCTTTCCACGATATCCTGGGCGTCrG 250 Pdgfra_1544_RNA CATCCAGTCGATTTCTGGAATCTACGAGTTTGCAGTCACGTGCGTCTATTTAGTGGAGCCTGTCCTATCTTCTTTTTCTTAATATGCTTGCAGArU

TABLE-US-00011 TABLE 11 List of PCR index primers used for monomer sequencing library preparation. SEQ ID NO: Name Sequence (5′-3) 251 fwd index1 AATGATACGGCGACCACCGAGATCTACACAGGCTATAACACTCTTTCCCTACACGAC 252 fwd index2 AATGATACGGCGACCACCGAGATCTACACGCCTCTATACACTCTTTCCCTACACGAC 253 fwd index3 AATGATACGGCGACCACCGAGATCTACACAGGATAGGACACTCTTTCCCTACACGAC 254 fwd index4 AATGATACGGCGACCACCGAGATCTACACTCAGAGCCACACTCTTTCCCTACACGAC 255 fwd index5 AATGATACGGCGACCACCGAGATCTACACCTTCGCCTACACTCTTTCCCTACACGAC 256 fwd index6 AATGATACGGCGACCACCGAGATCTACACTAAGATTAACACTCTTTCCCTACACGAC 257 fwd index? AATGATACGGCGACCACCGAGATCTACACACGTCCTGACACTCTTTCCCTACACGAC 258 fwd index8 AATGATACGGCGACCACCGAGATCTACACGTCAGTACACACTCTTTCCCTACACGAC 259 rev index1 CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGT 260 rev index2 CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGT 261 rev index3 CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTGGAGTTCAGACGTGT 262 P5primer AATGATACGGCGACCACCGA 263 P7primer CAAGCAGAAGACGGCATACGA bolded: illumina index sequences used to differentiate monomers in pooled samples; underlined: highlighted region indicating primer hybridisation site to the extended monomer.

TABLE-US-00012 TABLE 12 SEQ ID NO: Name Sequence (5′.fwdarw.3′) 264 R_KRAS. AGCTCCAACTACCACAAAGTCGArUAGTCACGGCTACTCArGACGTAACGCGTTCAGrUGATGCCCTTGCCTAC D3 GCCACC 265 R_KRAS. AGCTCCAACTACCACAArAGTCGArUAGTCArCGGCTArCTCAGArCGTAACGrCGTTCAGrUGATGCrCCTTGCCT D8 ACGCCACC

TABLE-US-00013 TABLE 13 267 R_KRAS.2G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 268 R_KRAS.2C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 269 R_KRAS.2A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArACGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 270 R_KRAS.2U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 271 R_KRAS.3G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGrGTTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 272 R_KRAS.3C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCrCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 273 R_KRAS.3A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArArAGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 274 R_KRAS.3U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUrUCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 275 R_KRAS.3 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 276 R_KRAS.2 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrACGTAACGCGTTCAGTGAT GCCCTTGCCTACGCCACC 277 R_KRAS Wt1_2 AGCTCCAACTACCACAA[1]CArGrACGTAACGCGTTCAGTGATGCCCTTGCCTACGCCACC 278 R_KRAS Wt1_3 AGCTCCAACTACCACAA[1]CArGrArCGTAACGCGTTCAGTGATGCCCTTGCCTACGCCACC 279 R_KRAS Wt1_4 AGCTCCAACTACCACAA[1]CArGrArCrGTAAC GCGTTCAGTGATGCCCTTGCCTACGCCACC 280 R_KRAS wt1_5 AGCTCCAACTACCACAA[1]CArGrArCrGrUAA CGCGTTCAGTGATGCCCTTGCCTACGCCACC 281 R_KRAS wt1_7 AGCTCCAACTACCACAA[1]CArGrArCrGrUrA rACGCGTTCAGTGATGCCCTTGCCTACGCCACC SEQ ID NO: 266: Bacteriophage Phi29 WT polymerase MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMG QWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLK GFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFEL KEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGK FASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFK RAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK