Method of immobilising RNA onto a surface

Abstract

The invention relates to a method of immobilising at least one RNA molecule onto a surface of a support comprising: i) providing a first support having a surface on which at least one DNA molecule is immobilised, wherein the DNA molecule encodes an RNA molecule and the encoded RNA molecule comprises a binding molecule; ii) providing a second support having a surface on which at least one binding partner for interacting with the binding molecule is immobilised; iii) arranging the first and second supports such that the surfaces displaying the immobilised molecules are in close proximity and substantially face each other, and contacting the DNA molecule immobilised on the surface of the first support with transcription reagents; and iv) carrying out a transcription reaction to generate the encoded RNA molecule, wherein the RNA molecule is directly immobilised onto the surface of the second support via an interaction between the binding molecule of the RNA molecule and the binding partner on the surface of the second support.

Claims

1. A method of immobilising a plurality of RNA molecules onto a surface of a support comprising: i) providing a first support that is a continuous planar surface on which a plurality of DNA molecules are immobilised, wherein each DNA molecule encodes an RNA molecule comprising a binding molecule; ii) providing a second support that is a continuous planar surface on which a plurality of binding partners for interacting with the binding molecules are immobilised; iii) arranging the first and second supports such that the surfaces displaying the immobilised DNA molecules and binding partners are in close proximity and substantially face each other, and contacting the DNA molecules immobilised on the surface of the first support with transcription reagents such that the surfaces of the first and second support are in contact with the transcription reagents; and iv) carrying out a transcription reaction between the first and second support to generate the RNA molecules, wherein the RNA molecules are directly immobilised onto the surface of the second support via an interaction between the binding molecule of the RNA molecule and the binding partner on the surface of the second support.

2. The method according to claim 1, wherein the DNA molecules comprise a promoter sequence operably linked to a sequence encoding the RNA molecule.

3. The method according to claim 2, wherein the promoter sequence is specific for T7 RNA polymerase.

4. The method according to claim 1, wherein the binding molecule is an RNA aptamer.

5. The method according to claim 4, wherein the RNA aptamer is a tobramycin-binding RNA aptamer or a streptavidin-binding RNA aptamer.

6. The method according to claim 5, wherein the binding partner immobilised on the surface of the second support is tobramycin or streptavidin.

7. The method according to claim 1, wherein the DNA molecule is immobilised onto the surface of the first support using biotin and streptavidin.

8. The method according to claim 1, wherein the plurality of DNA molecules have the same sequences such that the RNA molecules have the same sequences.

9. The method according to claim 1, wherein the plurality of DNA molecules have different sequences such that the RNA molecules have different sequences.

10. The method according to claim 1, wherein the RNA molecules encoded by the plurality of DNA molecules comprise the same binding molecule.

11. The method according to claim 1, wherein the RNA molecules encoded by the plurality of DNA molecules comprise different binding molecules.

12. The method according to claim 1, wherein the plurality of binding partners are the same.

13. The method according to claim 1, wherein the plurality of binding partners are different.

14. The method according to claim 1, wherein the first support is in an array format.

15. The method according to claim 1, wherein the second support is in an array format.

16. The method according to claim 1, wherein the step of arranging the first and second supports further comprises providing at least one spacing element to separate the surfaces of the first and second support such that the surfaces are not in direct contact.

17. A kit for carrying out the method claim 1, comprising: i) a first support that is a continuous planar surface comprising a plurality of DNA molecules immobilised thereon, wherein each DNA molecule encodes an RNA molecule comprising a binding molecule; ii) a second support that is a continuous planar surface for immobilising the RNA molecules encoded by the plurality of DNA molecules thereon, wherein a plurality of binding partners for interacting with the binding molecules are immobilised on the surface of the second support; and iii) transcription reagents, wherein the first and second supports can be arranged such that the surfaces displaying the immobilised DNA molecules and binding partners are in close proximity and substantially face each other.

18. The kit according to claim 17, further comprising means for securing the first and second supports such that the surfaces displaying the immobilised DNA molecules and binding partners are in close proximity and substantially face each other.

19. The kit according to claim 17, wherein the first support is in an array format.

20. The kit according to claim 17, wherein the second support is in an array format.

21. The kit according to claim 17, further comprising at least one spacing element for separating the surfaces of the first and second support such that, in use, the surfaces are not in direct contact.

22. The method according to claim 1, wherein the binding molecule is a nucleotide sequence and the binding partner is an oligonucleotide that is complementary to the nucleotide sequence of the binding molecule.

23. The method according to claim 1, wherein the method further comprises translating the immobilized RNA molecules to produce protein molecules.

24. The method according to claim 23, wherein the protein molecules are immobilized onto a third support following translation.

25. The kit according to claim 17, further comprising a third support having a surface for immobilizing the protein molecules encoded by the RNA molecules thereon.

26. The kit according to claim 25, further comprising translation agents.

27. The method according to claim 1, wherein the binding molecule is located at the 3′ end of the RNA molecule such that only the full length RNA molecule comprises the binding molecule.

28. The method according to claim 1, wherein the surfaces of the first and second supports are separated by a gap of about 10-200 μm.

29. The method according to claim 1, wherein the DNA molecules immobilised on the surface of the first support are contacted with transcription reagents such that the surfaces of the first and second support are in contact with the transcription reagents in a single continuous transcription mix.

Description

(1) The invention will now be described in detail by way of examples only with reference to the following figures:

(2) FIG. 1. ‘Sandwich print’ set-up arrangement. A schematic diagram is shown illustrating the sandwich arrangement of the DNA-template slide, with immobilised DNA template encoding the RNA of interest, +/− linker, with a specific tag (e.g. tobramycin aptamer (TobApt), streptavadin aptamer (SAApt), poly-A sequence (Atail)). The tag provides a means of enabling the subsequently synthesised RNA to bind to surface-immobilised binding-partner molecules (e.g. tobramycin, streptavadin, poly-dT) immobilised on the RNA-binding slide facing the DNA-template slide. The slides are sandwiched such that the DNA and RNA-binding molecules are aligned, although in some situations the RNA-binding slide can be completely coated in the RNA-binding molecule, removing the need for alignment. In all cases, the DNA-template and RNA-binding slides both face inwards, with the in vitro transcription mix in between. A small piece of parafilm at the ends of the slides is used as a spacer to prevent the slide surfaces from coming into direct contact.

(3) FIG. 2. Example gel showing that biotin-tagged DNA template is pure. Shown is a photograph of an 1.2% agarose gel following electrophoresis of PCR synthesised EV71-IRES.sub.TobApt DNA. The DNA was stained with ethidium bromide and visualised under UV. The successful synthesis of the Biotin-DNA template at high purity is seen by the band on the gel of the correct size.

(4) FIG. 3. EV71-IRES.sub.TobApt RNA is successfully ‘sandwich printed’ onto a tobramycin RNA-binding slide. Shown is a photograph of a tobramycin slide following ‘sandwich printing’ of the RNA molecule, EV71-IRES.sub.TobApt, synthesised from transcription of Biotin-EV71-IRES.sub.TobApt. The RNA molecules bound to the tobramycin were stained with SYBR gold and visualized under UV.

(5) FIG. 4. ‘Sandwich printed’ EV71-IRES.sub.TobApt RNA binds stably to the tobramycin RNA-binding slide. Shown is a photograph of the tobramycin slide from FIG. 3 following 3 washes with PBS buffer. EV71-IRES.sub.TobApt RNA is stably bound to tobramycin as it is still detected following 3 washes with PBS.

(6) FIG. 5. ‘Sandwich printed’ tobramycin apatmer (TobApt)-tagged RNAs are bound specifically to the tobramycin RNA-binding slide. RNAs HapR+/−TobApt and M-S+/−TobApt were ‘sandwich printed’ from their corresponding DNA template, in a four spot array format, on the DNA template slide, onto the tobramycin RNA-binding slide opposite. Cy.sup.5-labelled UTP was included in the in vitro transcription mix resulting in the RNAs produced being Cy.sup.5-labelled. Shown is the tobramycin RNA-binding slide, visualised at 639 nm for Cy.sup.5. Only the HapR.sub.TobApt and M-S.sub.TobApt bound to the tobramycin, as seen by the spots. The non-TobApt-RNAs failed to bind.

(7) FIG. 6. As for FIG. 5, except the RNAs used were Qrr1+/−TobApt and MicA+/−TobApt. Cy.sup.3-labelled UTP was included in the in vitro transcription mix resulting in the RNAs produced being Cy.sup.3-labelled. Shown is the tobramycin RNA-binding slide, visualised at 532 nm for Cy.sup.3. Only the Qrr1.sub.TobApt and MicA.sub.TobApt bound to the tobramycin, as seen by the spots. The non-TobApt-RNAs failed to bind.

(8) FIG. 7. TobApt-RNAs of a range of sizes can be immobilised using the ‘sandwich print’ method. Shown is a tobramycin RNA-binding slide following ‘sandwich printing’ with EV71-IRES.sub.TobApt and U1.sub.TobApt RNAs of 623 and 25 nucleotides respectively (with linkers of 83 and 20 nucleotides respectively and TobApt of 40 nucleotides). Cy.sup.3 UTP was incorporated in the in vitro transcription and the slide visualised at 532 nm showing the immobilised RNAs as spots.

(9) FIG. 8. ‘Sandwich printed’ streptavidin apatmer (SAApt)-tagged RNAs are bound specifically to the streptavidin RNA-binding slide. RNA MicA+/−SAApt was ‘sandwich printed’ from its corresponding DNA template on the DNA-template slide onto the streptavidin RNA-binding slide opposite. Cy.sup.3-labelled UTP was included in the in vitro transcription mix, between the slides, resulting in the RNA produced being Cy.sup.3-labelled. Shown is the streptavidin RNA-binding slide, visualised at 532 nm for Cy.sup.3. Only the MicA.sub.SAApt bound to the streptavidin RNA-binding slide, as seen by the spot. The non-SAApt-RNA (MicA) failed to bind.

(10) FIG. 9. Different SAApt-RNAs can be immobilised using the ‘sandwich print’ method. Shown is a streptavidin RNA-binding slide following ‘sandwich printing’ with U1.sub.SAApt and Qrr1.sub.SAApt RNAs of 25 and 99 nucleotides respectively (both with linkers of 26 nucleotides and SAApt of 44 nucleotides). Cy.sup.5 UTP was incorporated in the in vitro transcription for Qrr1.sub.SAApt and the slide visualised at 639 nm whereas Cy.sup.3 UTP was incorporated in the in vitro transcription for U1.sub.SAApt and the slide visualised at 532 nm. The ‘sandwich printed’ U1.sub.SAApt and Qrr1.sub.SAApt are seen as spots.

(11) FIG. 10. RNA's can be ‘sandwich printed’ on the same array slide via different RNA tags. RNAs M-S.sub.TobApt and M-S.sub.SAApt were ‘sandwich printed’ from their corresponding DNA template, in a two spot array format on the DNA template slide, onto an RNA-binding slide opposite spotted with the corresponding RNA binding molecules of tobramycin and streptavidin. Cy.sup.3-labelled UTP was included in the in vitro transcription mix resulting in the RNAs produced being Cy.sup.3-labelled. Shown is the tobramycin and streptavidin spotted RNA-binding slide, visualised at 532 nm for Cy.sup.3. Both M-S.sub.TobApt and M-S.sub.SAApt are seen by the spots.

(12) FIG. 11. RNAs transcribed using the ‘sandwich print’ method and incorporating a 15mer polyA-tail (Atail) can be immobilised to a poly-dT RNA-binding slide. Shown is a poly-dT RNA-binding slide following ‘sandwich printing’ of Qrr1.sub.Atail and M-S.sub.Atail RNAs of 91 and 68 nucleotides respectively (plus polyA-tail of 15 nucleotides respectively). Cy.sup.5 UTP was incorporated in the in vitro transcription reaction and the slide visualised at 639 nm. The bound RNA is seen as spots.

MATERIALS AND METHODS

(13) Preparation of DNA Templates

(14) EV71-IRES.sub.TobApt was PCR-amplified from pCRII IRES EV71 tob (Nicolas Locker, University of Surrey) using a biotinylated primer corresponding to the T7 promoter sequence (5′ biotin-ctc gag taa tac gac tca cta tag g 3′ (SEQ ID NO: 1), the T7 promoter sequence is in bold) and a primer complementary to the 3′ end of the EV71-IRES.sub.TobApt sequence (5′ agagagGGCTCAGCACGAGTGTAG 3′ (SEQ ID NO: 2), the region complementary to the 3′ end of the tobramycin aptamer sequence is in capitals). The DNA was cleaned up using the Nucleospin Extract II Kit (Macherey-Nagel, Düren, Germany).

(15) The EV71-IRES.sub.TobApt DNA template sequence is shown below:

(16) TABLE-US-00001 (SEQ ID NO: 3) Biotin-ctc gag taa tac gac tca cta taG GGA GAC GAT CAA TAG CAG GTG TGG CAC ACC AGT CAT ACC TTG ATC AAG CAC TTC TGT TTC CCC GGA CTG AGT ATC AAT AGG CTG CTC GCG CGG CTG AAG GAG AAA ACG TTC GTT ACC CGA CCA ACT ACT TCG AGA AGC TTA GTA CCA CCA TGA ACG AGG CAG GGT GTT TCG CTC AGC ACA ACC CCA GTG TAG ATC AGG CTG ATG AGT CAC TGC AAC CCC CAT GGG CGA CCA TGG CAG TGG CTG CGT TGG CGG CCT GCC CAT GGA GAA ATC CAT GGG ACG CTC TAA TTC TGA CAT GGT GTG AAG AGC CTA TTG AGC TAG CTG GTA GTC CTC CGG CCC CTG AAT GCG GCT AAT CCT AAC TGC GGA GCA CAT GCT CAC AAA CCA GTG GGT GGT GTG TCG TAA CGG GCA ACT CTG CAA CGG AAC CGA CTA CTT TGG GTG TCC CGT GTT TCC TTT TAT TCC TAT ATT GGC TGC TTA TGG TGA CAA TCA AAA AGT TGT TAC CAT ATA GCT ATT GGA TTG GCC ATC CGG TGT GCA ACA GGG CAA TTG TTT ACC TAT TTA TTG GTT TTG TAC CAT TAT CAC TGA AGT CTG TGA TCA CTC TCA AAT TCA TTT TGA CCC TCA ACA CAA TCA AAC atg agc acg aat cct aaa cct caa aga aaa acc aaa cgt aac acc aac cgt cgc cca caa acc tcg act ctt cta gac tct ctg gct tag tat agc gag gtt tag cta cac tcg tgc tga gcc ctc tct
(T7 promoter—bold; transcriptional start site—bold and underlined; EV71-IRES—capitals (623 nt); Linker—italics (83 nt); Tobramycin-binding aptamer (TobApt)—underlined (40 nt))

(17) All other DNA templates were generated by the extension of overlapping primers (Gao et al., (2003)). This was followed by PCR amplification using a biotinylated or thiolated primers corresponding to the T7 promoter sequence (5′ biotin/thiol-ctc gag taa tac gac tca cta tag g 3′ (SEQ ID NO: 1), the T7 promoter sequence is in bold) and a primer corresponding to the 3′ end of the required DNA sequence. This generated biotin/thiol-tagged DNA template for subsequent immobilisation to DNA-template slides. The DNA was cleaned up using the Nucleospin Extract II Kit (Macherey-Nagel, Düren, Germany).

(18) Below is a list of the sequences of the DNA templates prepared in this manner: 1. HapR

(19) TABLE-US-00002 (SEQ ID NO: 4) Biotin-ctc gag taa tac gac tca cta taG GGC TTT AAG TAG CAA ATA ACA AAA TAA TCA TTA GAG CAA AAT GCT CAA TCA ACA ACT CAA TTG GCA AGG ATA TAC CCC TAT GGA CGC AT  (T7 promoter—bold; Transcriptional start site—capitals and underlined; HapR—capitals (90 nt)). 2. MicA.sub.stab

(20) TABLE-US-00003 (SEQ ID NO: 5) Biotin-ctc gag taa tac gac tca cta taG AAA GAC GCG CAT TTG TTA TCA TCA TCC CTG GGA AAG CGA GGC TTT CCC TGG CCA CTC ACG AGT GGC CTT TT  (T7 promoter—bold; Transcriptional start site—capitals and underlined; MicA.sub.stab—capitals (71 nt)). 3. Qrr1

(21) TABLE-US-00004 (SEQ ID NO: 6) Biotin-ctc gag ta ata cga ctc act ata GGG TGA CCC GCA AGG GTC ACC TAG CCA ACT GAC GTT GTT AGT GAA TAA TCA ATG TTC ACA AAT AAC AGC CAA TAG ACT CAT TCT ATT GGC TAT TTT TTT  (T7 promoter—bold; Transcriptional start site—bold and underlined; Qrr1—capitals (99 nt)). 4. MicA

(22) TABLE-US-00005 (SEQ ID NO: 7) Biotin-ctc gag taa tac gac tca cta ta GGG GAA AGA CGC GCA TTT GTT ATC ATC ATC CCT GAA TTC AGA GAT GAA ATT TTG GCC ACT CAC GAG TGG CCT TTT  (T7 promoter—bold; Transcriptional start site—capital and underlined; MicA—capitals (75 nt)). 5. HapR.sub.TobApt

(23) TABLE-US-00006 (SEQ ID NO: 8) Biotin-ctc gag taa tac gac tca cta taG GGC TTT AAG TAG CAA ATA ACA AAA TAA TCA TTA GAG CAA AAT GCT CAA TCA ACA ACT CAA TTG GCA AGG ATA TAC CCC TAT GGA CGC AT a aaa aaa aaa aaa aaa aaa ctt agt ata gcg agg ttt agc tac act cgt gct gag cc  (T7 promoter—bold; Transcriptional start site—capitals and underlined; HapR—capitals (90 nt); Linker—italics (19 nt); Tobramycin aptamer—lower case, underlined (38 nt)). 6. HapR-no linker.sub.TobApt

(24) TABLE-US-00007 (SEQ ID NO: 9) Biotin-ctc gag taa tac gac tca cta taG GGC TTT AAG TAG CAA ATA ACA AAA TAA TCA TTA GAG CAA AAT GCT CAA TCA ACA ACT CAA TTG GCA AGG ATA TAC CCC TAT GGA CGC AT ctt agt ata gcg agg ttt agc tac act cgt gct gag cc  (T7 promoter—bold; Transcriptional start site—capitals and underlined; HapR—capitals (90 nt); Tobramycin aptamer—lower case, underlined (38 nt)) 7. M-S.sub.TobApt

(25) TABLE-US-00008 (SEQ ID NO: 10) Biotin-ctc gag taa tac gac tca cta taG AAA GAC GCG CAT TTG TTA TCA TCA TCC CTG GGA AAG CGA GGC TTT CCC TGG CCA CTC ACG AGT GGC CTT TT ata tcc ccc ccc ccc ccc cc ggc tta gta tag cga ggt tta gct  aca ctc gtg ctg agc c  (T7 promoter—bold; Transcriptional start site—capital and underlined; M-S—capitals (71 nt); Linker—italics (20 nt); Tobramycin binding aptamer.sub.(TobApt)—underlined (40 nt)). 8. Qrr1.sub.TobApt

(26) TABLE-US-00009 (SEQ ID NO: 11) Biotin-ctc gag ta ata cga ctc act ata GGG TGA CCC GCA AGG GTC ACC TAG CCA ACT GAC GTT GTT AGT GAA TAA TCA ATG TTC ACA AAT AAC AGC CAA TAG ACT CAT TCT ATT GGC TAT TTT TTT ttt ttt ttt tcc ccc ccc cc g gct tag tat agc gag gtt tag cta cac tcg tgc tga gcc  (T7 promoter—bold; Transcriptional start site—capitals and underlined; Qrr1—capitals (99 nt); Linker—italics (20 nt); Tobramycin binding aptamer.sub.(TobApt)—underlined (40 nt)). 9. MicA.sub.TobApt

(27) TABLE-US-00010 (SEQ ID NO: 12) Biotin-ctc gag taa tac gac tca cta ta GGG GAA AGA CGC GCA TTT GTT ATC ATC ATC CCT GAA TTC AGA GAT GAA ATT TTG GCC ACT CAC GAG TGG CCT TTT aca cac aca cac aca cac ac ggc tta gta tag cga ggt tta gct aca ctc gtg ctg agc c  (T7 promoter—bold; Transcriptional start site—capitals and underlined; MicA—capitals (75 nt); Linker—italics (20 nt); Tobramycin binding aptamer.sub.(TobApt)—underlined (40 nt)). 10. U1.sub.TobApt

(28) TABLE-US-00011 (SEQ ID NO: 13) Biotin-ctc gag taa tac gac tca cta taG GG TAT CCA TTG CAC TCC GGA TGC C ttt ttt ttt tcc ccc ccc cc g gct tag tat agc gag gtt tag cta cac tcg tcg  tga gcc  (T7 promoter—bold; Transcriptional start site—capitals and underlined; U1—capitals (25 nt); Linker—italics (20 nt); Tobramycin binding aptamer.sub.(TobApt)—underlined (40 nt)). 11. Qrr1.sub.SAApt

(29) TABLE-US-00012 (SEQ ID NO: 14) Biotin-ctc gag ta ata cga ctc act ata GGG TGA CCC GCA AGG GTC ACC TAG CCA ACT GAC GTT GTT AGT GAA TAA TCA ATG TTC ACA AAT AAC AGC CAA TAG ACT CAT TCT ATT GGC TAT TTT TTT ttt ttt ttt ttt ttt ttt ttt gtg tg acc gac cag aat cat gca agt gcg taa gat agt cgc ggg ccg gg cac aca  (T7 promoter—bold; Transcriptional start site—capitals and underlined; Qrr1—capitals (99 nt); Linker—italics (26 nt); Streptavidin binding aptamer.sub.(SAApt)—underlined (44 nt); Linker 2—italics underlined (6 nt)). 12. M-S.sub.SAApt

(30) TABLE-US-00013 (SEQ ID NO: 15) Biotin-ctc gag taa tac gac tca cta taG AAA GAC GCG CAT TTG TTA TCA TCA TCC CTG GGA AAG CGA GGC TTT CCC TGG CCA CTC ACG AGT GGC CTT TT aca cac aca cac aca cac acg cat gca t acc gac cag aat cat gca agt gcg taa gat agt cgc ggg ccg gg atg cat gc  (T7 promoter—bold; Transcriptional start site—capital and underlined; MicA.sub.stab—capitals (72 nt); Linker—italics (28 nt); Streptavidin binding aptamer.sub.(SAApt)—underlined (44 nt); Linker 2—italics underlined (8nt)). 13. MicA.sub.SAApt

(31) TABLE-US-00014 (SEQ ID NO: 16) Biotin-ctc gag taa tac gac tca cta ta GGG GAA AGA CGC GCA TTT GTT ATC ATC ATC CCT GAA TTC AGA GAT GAA ATT TTG GCC ACT CAC GAG TGG CCT TTT aca cac aca cac aca cac acg cat gca t acc gac cag aat cat gca agt gcg taa gat agt cgc ggg ccg gg atg cat gc  (T7 promoter—bold; Transcriptional start site—capitals and underlined; MicA—capitals (75 nt); Linker—italics (28 nt); Streptavidin binding aptamer.sub.(SAApt)—underlined (44 nt); Linker 2—italics underlined (8 nt)). 14. U1.sub.SAApt

(32) TABLE-US-00015 (SEQ ID NO: 17) Biotin-ctc gag taa tac gac tca cta taG GGT ATC CAT TGC ACT CCG GAT GCC ttt ttt ttt ttt ttt ttt ttt gtg tg acc gac cag aat cat gca agt gcg taa gat agt cgc ggg ccg gg cac aca  (T7 promoter—bold; Transcriptional start site—capitals and underlined; U1—capitals (25 nt); Linker—italics (26 nt); Streptavidin binding aptamer.sub.(SAApt)—underlined (44 nt); Linker 2—italics underlined (6 nt)). 15. Qrr1.sub.Atail

(33) TABLE-US-00016 (SEQ ID NO: 18) Biotin-ctc ta ata cga ctc act ata GGG TGA CCC GCA AGG GTC ACC TAG CCA ACT GAC GTT GTT AGT GAA TAA TCA ATG TTC ACA AAT AAC AGC CAA TAG ACT CAT TCT ATT GGC T aaa aaa aaa aaa aaa  (T7 promoter—bold; Transcriptional start site—capitals and underlined; Qrr1—capitals (91 nt); polyA.sub.(A tail)—italics (15 nt)). 16. M-S.sub.Atail

(34) TABLE-US-00017 (SEQ ID NO: 19) Biotin-ctc gag taa tac gac tca cta taG AAA GAC GCG CAT TTG TTA TCA TCA TCC CTG GGA AAG CGA GGC TTT CCC TGG CCA CTC ACG AGT GGC C aaa aaa aaa aaa aaa  (T7 promoter—bold; Transcriptional start site—capital and underlined; MicA.sub.stab—capitals (68 nt); polyA.sub.(A tail)—italics (15 nt)).
Preparation of DNA-Template Slides

(35) Using either home-prepared streptavidin-spotted slides (see below) or commercially available streptavidin-coated slides (Microsurfaces), ˜10 μl of 200 nM Biotin-DNA template in phosphate-buffered saline (PBS) was spotted onto the streptavidin. Slides were incubated at 37° C. in a humidified petri dish for ˜30 minutes. Slides were washed ˜3× with ˜5 ml PBST (PBS with 0.5% Tween), ˜1× with H.sub.2O and air dried.

(36) Alternatively, NHS-activated slides were treated with 80 mM PDEA in 0.1M sodium borate pH8.5 for 30 minutes to produce reactive disulphide groups. ˜10 μl of 200 nM Thiol-DNA template in phosphate-buffered saline (PBS) was spotted onto the activated slide. Slides were incubated at 37° C. in a humidified petri dish for ˜30 minutes. Slides were washed ˜3× with ˜5 ml PBST (PBS with 0.5% Tween), ˜1× with H.sub.2O and treated with 50 mM cysteine and 1 M NaCl in 0.1 M sodium acetate pH 4 for ˜30 minutes to deactivate excess reactive groups.

(37) Preparation of RNA-Binding Slides

(38) 1) Immobilization of Tobramycin or Streptavidin to Slides

(39) ˜10 μl spots of 5 mM tobramycin or 16.6 μM streptavadin in PBS were pipetted onto NHS-activated slides (Schott Nexterion Slide H). The slides were incubated at 37° C. in a humidified petri dish for ˜1 hour. The slides were washed ˜3× with ˜5 ml PBST (0.5% Tween) and ˜1× with H.sub.2O. The remainder of the NHS-activated surface was blocked with ethanolamine. ˜5 ml of 50 mM ethanolamine-HCl was used to cover the slides and they were incubated at room temperature for ˜1 hr. The slides were washed ˜3× with ˜5 ml PBST (0.5% Tween), 1× with H.sub.2O and dried in air. 2) Immobilization of Poly-dT

(40) 25-mer poly-dT (Invitrogen) was chemically synthesised with a 5′ Biotin, re-suspended in PBS at 10 μM and aliquots slide-immobilised in the same way as for the Biotin-DNA templates, detailed above.

(41) RNA Synthesis Using the ‘Sandwich Print Set-Up’ and Subsequent Slide Visualisation

(42) 150 μl of MegaScript T7 in vitro transcription mix (Applied Biosystems, California, USA), sometimes including 0.05 mM Cy3 or Cy5 UTP to Cy-label the RNA, was pipetted over the RNA-binding slide. The DNA-template slide was then placed on top so that the spots of RNA-binding molecule and DNA-template were lined up (although the RNA-binding slides which are completely coated with RNA-binding molecule immobilised do not require specific alignment). In all cases the DNA-template and RNA-binding slides both face inwards. A small piece of parafilm at the ends of the slides was used as a spacer to prevent the slide surfaces from coming into direct contact. The arrangement of the two slides is shown in FIG. 1.

(43) Following incubation at 37° C. for ˜1-4 hrs, the slides were separated and the in vitro transcription mix was recovered from the slide surface using a pipette. The DNA template slide was washed ˜3× with ˜5 ml PBS, ˜1× with ˜5 ml H.sub.2O and air dried. The RNA-binding slide was washed ˜3× with ˜5 ml PBS, ˜1ט5 ml H.sub.2O and air dried. Cy-labelled RNA was visualized at 532 nm for Cy3 or 639 nm for Cy5. For unlabelled RNA, the RNA-binding slide was stained by covering the slide with ˜5 ml SYBR gold (Invitrogen, Paisley, UK; 5 μl of SYBR gold in ˜25 ml PBS) for 10 minutes and visualised with a UV transilluminator.

Results

(44) ‘Sandwich Print’ Set-Up

(45) The general experimental set-up arrangement used to conduct the ‘sandwich print’ studies is as shown in FIG. 1. RNAs containing different RNA-tags were synthesised from the DNA templates on the DNA-template slide. Through these RNA-tags, the synthesised RNAs bind to the RNA-binding molecules immobilised to the RNA-binding slide directly facing the DNA-template slide. For example, when the tobramycin aptamer was used as the RNA-tag, corresponding biotin-DNA template was immobilised in spots on a streptavidin-slide to create the DNA-template slide whilst tobramycin was covalently immobilised to an NHS-activated slide in spots to create the RNA-binding slide. The two slides were set-up to face each other, with the DNA-template spots aligned with the tobramycin spots (although the RNA-binding slide can be completely tobramycin-coated, removing the need for such alignment). Alternatively, when the streptavidin aptamer was used as the RNA-tag, the DNA-template slide was created by immobilising thiolated-DNA template to a NHS-activated slide in spot format or immobilising biotin-DNA template to streptavidin-spotted slides. For the RNA-binding slide, streptavidin spotted or coated slides were used and set-up facing the DNA-template slide, spots aligned as required. Similarly, when the polyA-tail was used as the RNA-tag, corresponding biotin-DNA template was immobilised in spots on a streptavidin slide to create the DNA-template slide. The RNA-binding molecule, Biotin-poly-dT (25mer), was similarly immobilised to a streptavidin slide to create the RNA-binding slide. The two slides were similarly arranged facing each other with spots aligned as required.

(46) Preparation of DNA Templates

(47) Agarose gel electrophoresis stained with ethidium bromide was used to analyse the DNA templates prior to slide-immobilisation to confirm products of the correct size had been synthesised. FIG. 2 shows an example of successful synthesis of a DNA template, namely, Biotin-EV71-IRES.sub.TobApt DNA template.

(48) RNA Synthesis (Sandwich Printing)

(49) The inventor confirmed that RNA of the correct length had been synthesized during the ‘sandwich print’ process by urea polyacrylamide gel electrophoresis of the ‘sandwich print’ in vitro transcription solution following incubation for 1-4 hours.

(50) Confirmation of successful RNA ‘sandwich printing’ is shown in FIG. 3 where EV71-IRES.sub.TobApt RNA, synthesized from immobilised Biotin-EV71-IRES.sub.TobApt DNA (immobilized on the streptavidin coated DNA slide), bound to immobilised tobramycin (on the facing RNA-binding slide). The RNA was stained with SYBR gold and visualized under UV. Tobramycin-bound EV71-IRES.sub.TobApt RNA was still detected following 3 washes with PBS (FIG. 4), indicating the RNA to be stably bound to the tobramycin surface. EV71-IRES lacking the tobramycin aptamer failed to bind to the tobramycin slide.

(51) To demonstrate that this method is successful for a range of RNA molecules of varying sizes and functions, a selection of RNA molecules, with and without tobramycin aptamers, were tested for ‘sandwich printing’ in a four spot array format. The mRNA, HapR, and small non-coding RNAs (sRNAs), Qrr1, MicA, as well as a mutated sRNA, M-S, were tested. These RNAs range from ˜75-100 nt in size, each with a linker of ˜20 nt and TobApt of 40 nt. Each RNA only bound to the tobramycin RNA-binding slide when incorporating the tobramycin aptamer, with the control RNAs, lacking the tobramycin aptamer, failing to bind (FIGS. 5 and 6). Similarly, the 623 nt large EV71-IRES.sub.TobApt RNA with a 83 nt linker prior to the TobApt as well as the smaller 25 nt U1.sub.TobApt RNA with 20 nt linker prior to the TobApt were both seen to be bound to tobramycin following ‘sandwich printing’ (FIG. 7).

(52) Whilst a number of different linkers between the RNA of interest and RNA-tag have been used, HapR.sub.TobApt+/−a linker between the HapR and TobApt has also been tested (data not shown). Both HapR.sub.TobApt+/−a linker were seen to bind to the RNA-binding slide following ‘sandwich print’.

(53) To demonstrate that the ‘sandwich print’ method is applicable to any RNA aptamer interaction, RNA incorporating a streptavidin aptamer (SAApt) as the RNA-tag was tested for binding to a streptavidin RNA-binding slide. FIG. 8 shows the RNA MicA+/−streptavadin aptamer following ‘sandwich print’. Only the MicA.sub.SAApt, incorporating the stretpavadin aptamer, bound to the streptavidin RNA-binding slide. The MicA control, lacking the aptamer, failed to bind. RNAs U1.sub.SAApt and Qrr1.sub.SAApt, of 25 and 99 nt respectively, both incorporating a 26 nt linker and 44 nt streptavidin aptamer, bound to the streptavidin RNA-binding slide following ‘sandwich print’ (FIG. 9).

(54) To demonstrate that RNA's can be ‘sandwich printed’ on the same array slide via different RNA tags, RNAs incorporating either a TobApt or SAApt were ‘sandwich printed’ from their corresponding DNA template onto an RNA-binding slide opposite spotted with the corresponding RNA binding molecules of tobramycin and streptavidin. FIG. 10 shows the RNAs M-S.sub.TobApt and M-S.sub.SAApt following ‘sandwich print’. The M-S.sub.TobApt bound to the tobramycin spot of the RNA-binding slide whilst the M-S.sub.SAApt bound to the streptavidin spot of the RNA-binding slide.

(55) Whilst RNA aptamers represent one form of RNA-tag that will bind tightly to a specific partner molecule, a complementary RNA-DNA base-pairing interaction can also be used to specifically bind RNA to a RNA-binding slide. To demonstrate this, the RNAs Qrr1 and M-S with 15-mer polyA tails were tested using the ‘sandwich print’ method for binding to immobilised 25mer poly-dT on the RNA-binding slide. FIG. 11 shows successful binding of the RNAs to the poly-dT RNA-binding slide.

REFERENCES

(56) Gao et al., (2003) Nucleic Acids Research, 31, e143 Kim et al., (2006) JACS 128, 12076-12077 Lee et al., (2006) (Corn group) Langmuir 22, 5241-5250 Sendroiu et al. (2011) Journal of the American Chemical Society, 133, 4271-4273; Wahlestedt C. (2006) Drug Discovery Today, 11, 503-08