RNA TEMPLATED LIGATION
20230366010 · 2023-11-16
Inventors
Cpc classification
C12Q2525/121
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
C12Q2525/179
CHEMISTRY; METALLURGY
C12Q2525/121
CHEMISTRY; METALLURGY
C12Q1/6876
CHEMISTRY; METALLURGY
C12Q2525/179
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
International classification
C12N15/11
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
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. (canceled)
2. A method of detecting a target RNA molecule in situ in a cell or tissue sample, the method comprising: (a) contacting the cell or tissue sample with a first oligonucleotide and a second oligonucleotide to hybridize the first oligonucleotide and the second oligonucleotide to the target RNA molecule, wherein the first oligonucleotide comprises a first target binding site at a 5′ end, wherein the second oligonucleotide comprises (i) a second target binding site at a 3′ end, and (ii) a ribonucleotide at a 3′ terminus; and wherein a 5′ terminus of the first oligonucleotide and the 3′ terminus of the second oligonucleotide are adjacent to one another when hybridized to the target RNA molecule; (b) ligating the first and second oligonucleotides hybridized to the target RNA molecule to generate a circularized probe, wherein the 5′ terminus of the first oligonucleotide is ligated to the 3′ terminus of the second oligonucleotide, and wherein the 3′ terminus of the first oligonucleotide is ligated to the 5′ terminus of the second oligonucleotide; (c) amplifying the circularized probe by rolling circle amplification (RCA) to generate an RCA product; and (d) detecting the RCA product at in the cell or tissue sample, thereby detecting the target RNA molecule at the location in the cell or tissue sample.
3. The method of claim 2, wherein the second oligonucleotide comprises at least one additional ribonucleotide, but no more than 4 consecutive ribonucleotides.
4. The method of claim 3, wherein two consecutive ribonucleotides are present at the 3′ terminus of the second oligonucleotide.
5. The method of claim 2, wherein the second oligonucleotide does not comprise any additional ribonucleotides.
6. The method of claim 2, wherein the first oligonucleotide comprises a ribonucleotide at the 3′ terminus.
7. The method of claim 2, wherein the first oligonucleotide comprises a deoxyribonucleotide comprising a 5′ phosphate group at the 5′ terminus.
8. The method of claim 2, wherein the 5′ terminus of the first oligonucleotide does not comprise a ribonucleotide.
9. The method of claim 2, wherein the first and second oligonucleotides are ligated without gap-fill extension or hybridization of a gap oligonucleotide to the target RNA molecule to fill a gap between the 5′ terminus of the first oligonucleotide and the 3′ terminus of the second oligonucleotide.
10. The method of claim 2, wherein ligating the first and second oligonucleotides comprises using a DNA/RNA ligase.
11. The method of claim 10, wherein the DNA/RNA ligase is an RNA ligase.
12. The method of claim 10, wherein the DNA/RNA ligase is T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, PBCV-1 ligase, or DraRN1 ligase.
13. The method of claim 2, further comprising contacting the cell or tissue sample with a third oligonucleotide, wherein the 3′ end of the first oligonucleotide and the 5′ end of the second oligonucleotide hybridize to the third oligonucleotide and are ligated using the third oligonucleotide as a ligation template.
14. The method of claim 13, wherein the cell or tissue sample is contacted with the third oligonucleotide subsequent to (a).
15. The method of claim 13, wherein the third oligonucleotide is prehybridized to the first oligonucleotide and the second oligonucleotide prior to (a).
16. The method of claim 13, wherein the first and second oligonucleotides are ligated without gap-fill extension or hybridization of a gap oligonucleotide to the third oligonucleotide to fill a gap between the 3′ end of the first oligonucleotide and the 5′ end of the second oligonucleotide.
17. The method of claim 13, wherein the first oligonucleotide comprises a 3′ terminal deoxyribonucleotide and the second oligonucleotide comprises a 5′ terminal deoxyribonucleotide.
18. The method of claim 17, wherein the third oligonucleotide is a DNA molecule.
19. The method of claim 18, wherein the 3′ terminus of the first oligonucleotide is ligated to the 5′ terminus of the second oligonucleotide using a DNA ligase.
20. The method of claim 19, wherein the 5′ terminus of the first oligonucleotide is ligated to the 3′ terminus of the second oligonucleotide using a DNA/RNA ligase.
21. The method of claim 20, wherein the DNA/RNA ligase is T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, PBCV-1 ligase, or DraRN1 ligase.
22. The method of claim 2, wherein the circularized probe comprises no more than 2 consecutive ribonucleotides.
23. The method of claim 2, wherein the RCA comprises using a Phi29 DNA polymerase.
24. The method of claim 2, wherein: (i) the first oligonucleotide comprises one or more further sequences selected from a barcode sequence or a primer binding sequence; (ii) the second oligonucleotide comprise one or more further sequences selected from a barcode sequence or a primer binding sequence; or (iii) the first oligonucleotide and the second oligonucleotide each comprise one or more further sequences selected from a barcode sequence or a primer binding sequence.
25. The method of claim 2, wherein (c) comprises extending a primer hybridized to the circularized probe to generate the RCA product.
26. The method of claim 2, wherein the first oligonucleotide or the second oligonucleotide comprise a primer binding sequence such that the circularized probe comprises the primer binding sequence and wherein (c) comprises hybridizing a primer to the circularized probe and extending the primer to generate the RCA product.
27. The method of claim 2, wherein (c) comprises using a 3′ portion of the target RNA molecule as a primer to generate the RCA product.
28. The method of claim 2, wherein the circularized probe comprises a barcode sequence that identifies the target RNA.
29. The method of claim 28, wherein (d) comprises hybridizing fluorescently labeled detection oligonucleotides to the RCA product at the barcode sequence and using imaging to detect fluorescent signals associated with the RCA product.
30. The method of claim 2, wherein at least 10, at least 20, at least 100, or at least 1,000 different target RNA sequences are detected in parallel or sequentially at locations in the cell or tissue sample.
31. The method of claim 2, wherein the cell or tissue sample is a tissue section on a solid support.
Description
[0222] The present invention may be better understood with reference to the Examples and Figures, in which:
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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
Material and Methods
Oligonucleotides Used in the Study
[0256] All oligonucleotides used were purchased from IDT (Integrated DNA Technologies, Inc., Coralville, IA, 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.
[0257] 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,
RNA Detection Assay and Digital Quantitation of Amplified iLock and Padlock Probes
[0258] 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 T4RnI2 (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.
Multiplexed miRNA Detection Using Chimeric iLock Probes and Sequencing-by Ligation
[0259] 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.
[0260] 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 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.
Results
Effect of 3′-OH RNA on PBCV-1 DNA Ligase RNA-Dependent Ligation: RNA End Joining for Different RNA Substrates
[0261] 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 T4RnI2 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 (
[0262] PBCV-1 ligase catalysed highly efficient end-joining when 3′ RNA containing PLPs were ligated on miRNA targets (
Effect of Various RNA Substitutions on RNA Templated iLock Probe Activation, Ligation Efficiency and Fidelity for PBCV-1 as Well as T4RnI2
[0263] 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 (
[0264] Compared to a non-chimeric let-7a iLock, iLock-3 greatly increased detection of let-7a miRNA. According to PAGE of ligated iLock probes (
Multiplexed Detection of Let-7 Isoforms Using Chimeric iLock Probes
[0265] 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 (
Example 2— Ligation of DNA Padlock Probes, and Chimeric Probes Comprising 1 or 2 Ribonucleotides at their 3′ End
Materials and Methods
[0266] 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.
Results
[0267] 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 (
[0268] On RNA templates the activity of Splint R ligase and T4 RNA ligase II are similar (
[0269] 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
Materials and Methods
[0270] 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 (
Results
[0271] Chimeric padlock probes resulted in a higher in situ RNA detection efficiency on both A549 and OncoDG1 cell lines compared to DNA padlock probes (
[0272] 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
Materials and Methods
[0273] In-Solution Gap-Fill iLock Reaction:
[0274] 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.
In Situ Gapfill iLock Reaction
[0275] 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.
Results
[0276] RCA products from in-solution gap-fill polymerization-invader reactions were quantified and plotted in in
Example 5—Use of Phi29 as a Reverse Transcriptase Enzyme
Materials and Methods
Oligonucleotides
[0277] 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.
[0278] 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.
[0279] 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.
Sequencing of RCA Products Generated from RNA Containing Circles
Monomerization
[0280] 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 Alul restriction enzyme in a reaction mixture containing 1×Phi29 DNA polymerase buffer, 2 mg/mL BSA, 100 nM restriction oligonucleotide (Alul KRAS RO—Table 9), 120 mU/μL Alul (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%.
Sequencing Library Preparation of RCA Monomers
[0281] 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.
Morphological Assessment of RCP Size and Intensity
[0282] 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.
Results
Phi29 DNA Polymerase Accepts Chimeric Circles as Rolling Circle Amplification (RCA) Templates
[0283] 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 (
[0284] 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.
[0285] 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 (
[0286] 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 (
[0287] We have observed no effect on the RCA efficiency when a single RNA was substituted in the probe (circle) backbone (
[0288] 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 (
Phi 29 DNA Polymerase Preferentially Reverse Transcribes RNA Pyrimidines During RCA
[0289] 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 (
[0290] 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 (
Manganese Ions Increase RNA-Dependent RCA Activity of Phi29 DNA Polymerase
[0291] 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 (
Sequencing of Rolling Circle Products Demonstrates Ability of Phi29 DNA Polymerase to Reverse Transcribes RNA
[0292] 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 (
[0293] 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
[0294] 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
[0295] 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.
[0296] 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
Materials and Methods
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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!.
Results
[0301] Though probes were pooled together, only the expected signal was observed in cells (
Example 7— Detection of miR21 on a Solid Support Using DNA PLPs, Chimeric PLPs, and DNA and Chimeric iLock Probes
[0302] 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.
Materials and Methods
[0303] 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.
[0304] 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×.
[0305] 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.
[0306] 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/μL 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.
[0307] Finally, decorator oligonucleotides were hybridised to RCA products at 0.1 pM 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.
Results
[0308] 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
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
Materials and Methods
[0309] 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 3h 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.
[0310] 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 2nd 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 2nd 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 3rd and 4th 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).
Results
[0311] 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 (
TABLE-US-00001 TABLE 1 SEQ 5′ ID modifi- NO: Name cation Sequence (5′-3′) 1 hsa_ rArArCrUrUrGrUrGrGrUrArGrUrU KRAS rGrGrArGrCrUrGrGrUrGrGrCrGrU rArGrGrCrArArGrArGrUrGrCrC 2 KRASwt_ Phos AGCTCCAACTACCAC(10A)AGTAGCCG PLP TGACTATCGACT(10A)CTTGCCTACGC CACC 3 hsa_ rUrGrArGrGrUrArGrGrArGrGrUrU let-7e rGrUrArUrArGrUrU 4 hsa_ rArGrArGrGrUrArGrUrArGrGrUrU let-7d rGrCrArUrArGrUrU 5 let7-a_ Phos CTACTACCTCA(10A)CCTCAATGCACA PLP_1 TGTTTGGCTCC(10A)AACTATACAAC 6 let7-f_ Phos CTACTACCTCA(10A)CCTCAATGCACA PLP_1 TGTTTGGCTCC(10A)AACTATACAAT 7 let7-e_ Phos CTCCTACCTCA(10A)CCTCAATGCACA PLP_1 TGTTTGGCTCC(10A)AACTATACAAC 8 let7-d_ Phos CTACTACCTCT(10A)CCTCAATGCACA PLP_1 CTGTTTGGCTCC(10A)AACTATGCAA 9 let7-a_ Phos CTACTACCTCA(10A)CCTCAATGCACA PLP_ GTTTTGGCTCC(10A)AACTATACAArC RNA_1 10 let7-f_ Phos CTACTACCTCA(10A)CCTCAATGCACA PLP_ TGTTTGGCTCC(10A)AACTATACAArU RNA_1 11 let7-e_ Phos CTCCTACCTCA(10A)CCTCAATGCACA PLP_ TGTTTGGCTCC(10A)AACTATACAArC RNA_1 12 let7-d_ Phos CTACTACCTCT(10A)CCTCAATGCACA PLP_ TGTTTGGCTCC(10A)AACTATGCAArC RNA_1 13 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rC templ_C rCrCrUrArUrArUrCrCrUrCrG 14 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rA templ_A rCrCrUrArUrArUrCrCrUrCrG 15 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rG templ_G rCrCrUrArUrArUrCrCrUrCrG 16 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rU templ_U rCrCrUrArUrArUrCrCrUrCrG 17 3′T_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGT 18 3′G_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGG 19 3′A_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGA 20 3′C_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGC 21 3′rT_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGrT 22 3′rG_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGrG 23 3′rA_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGrA 24 3′rC_ Phos ATGACAGCGAGA(10A)AGTAGCCGTGA PLP_2 CTATCGACT(10A)CGAGGATATAGGrC 25 Decorator Cy3 CCTCAATGCACATGTTTGGCTCCa probe_1 26 Decorator Cy3 AGTAGCCGTGACTATCGACTa probe2_2 r[N]: RNA oligonucleotide; (10A): linker; a: last four bases of the decorator probe were 2′ O-methylRNA to prevent oligo hydrolysis by Phi29 polymerase; italics: decorator sequence.
TABLE-US-00002 TABLE 2 SEQ 5′ ID modifi- NO: Name cation Sequence (5′-3′) 27 hsa_ rUrGrAGrGrUrArGrUrArGrGrUrU let-7a rGrUrArUrArGrUrU 28 let- Phos CTACTACCTCA(7A)CCTCAATGCACA 7a_PLP TGTTTGGCTCC(7A)AACTATACAAC 29 iLock_1 CGCGTGTCGTTGCCCTACTACCTCA (10A)CCTCAATGCACATGTTTGGCTCC (10A)AACTATACAAC 30 iLock- CGCGTGTCGTTGCCCTACTACCTCA 3_1 (10A)CCTCAATGCACATGTTTGGCTCC (10A)AACTATACAArC 31 iLock- CGCGTGTCGTTGCrCCTACTACCTCA 3D_1 (10A)CCTCAATGCACATGTTTGGCTCC (10A)AACTATACAArC 32 iLock- CGCGTGTCGTTGCrCrCTACTACCTCA 3D5_1 (10A)CCTCAATGCACATGTTTGGCTCC (10A)AACTATACAArC 33 iLock- rCrGrCrGrTrGrTrCrGrTrTrGrCrC 3DF_1 CTACTACCTCA(10A)CCTCAATGCACA TGTTTGGCTCC(10A)AACTATACAArC 34 iLock- rCrGrCrGrTrGrTrCrGrTrTrGrCrC DF_1 CTACTACCTCA(10A)CCTCAATGCACA TGTTTGGCTCC(10A)AACTATACAAC 35 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rCr templ_C CrCrUrArUrArUrCrCrUrCrG 36 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rAr templ_A CrCrUrArUrArUrCrCrUrCrG 37 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rGr templ_G CrCrUrArUrArUrCrCrUrCrG 38 benchm_ rUrCrUrCrGrCrUrGrUrCrArU*rUr templ_U CrCrUrArUrArUrCrCrUrCrG 39 3′T_ TATATCCCTATATTATGACAGCGAGA iLock_3 (10A)AGTAGCCGTGACTATCGACT (10A)CGAGGATATAGGT 40 3′G_ TATATCCCTATATGATGACAGCGAGA iLock_3 (10A)AGTAGCCGTGACTATCGACT (10A)CGAGGATATAGGG 41 3′A_ TATATCCCTATATAATGACAGCGAGA iLock_3 (10A)AGTAGCCGTGACTATCGACT (10A)CGAGGATATAGGA 42 3′C_ TATATCCCTATATCATGACAGCGAGA iLock_3 (10A)AGTAGCCGTGACTATCGACT (10A)CGAGGATATAGGC 43 3′U_ TATATCCCTATATrUATGACAGCGAGA iLock_ (10A)AGTAGCCGTGACTATCGACT RNA_3 (10A)CGAGGATATAGGrU 44 3′G_ TATATCCCTATATrGATGACAGCGAGA iLock_ (10A)AGTAGCCGTGACTATCGACT RNA_3 (10A)CGAGGATATAGGrG 45 3′A_ TATATCCCTATATrAATGACAGCGAGA iLock_ G(10A)AGTAGCCGTGACTATCGACT RNA_3 (10A)CAGGATATAGGrA 46 3′C_ TATATCCCTATATrCATGACAGCGAGA iLock_ (10A)AGTAGCCGTGACTATCGACT RNA_3 (10A)CGAGGATATAGGrC 47 Decor- Cy3 CCTCAATGCACATGTTTGGCTCCa ator probe_ 1 48 Decor- Cy3 TGCGTCTATTTAGTGGAGCCa ator probe_ 2 49 Decor- Cy3 AGTAGCCGTGACTATCGACTa ator probe_ 3
TABLE-US-00003 TABLE 3 SEQ ID NO: Name Sequence (5′-3′) 50 hsa_ rUrGrAGrGrUrArGrUrArGrGrUrUrGrU let-7a rArUrArGrUrU 51 hsa_ rUrGrArGrGrUrArGrUrArGrArUrUrGrU let-7f rArUrArGrUrU 52 hsa_ rUrGrArGrGrUrArGrGrArGrGrUrUrGrU let-7e rArUrArGrUrU 53 hsa_ rArGrArGrGrUrArGrUrArGrGrUrUrGrC let-7d rArUrArGrUrU 54 let-7a_ AAAGATGCGATACrACTACCTCATGCGTCTAT seqRNA TTAGTGGAGCCCGCTATCTTCTTTAACTATAC AACCTrA 55 let-7e_ AAAATGTCGTTGCrCCTCCTACCTCATGCGTC seqRNA TATTTAGTGGAGCCGCCTATCTTCTTTAACTA TACAArC 56 let-7d_ AAAATGTCGTTGCrCCTACTACCTCTTGCGTC seqRNA TATTTAGTGGAGCCATCTATCTTCTTTAACTA TgCAArC 57 let-7f_ AAAATGTCGTTGCrUCTACTACCTCATGCGTC seqRNA TATTTAGTGGAGCCTACTATCTTCTTTAACTA TACAArU 58 let- AAAGATGCGATACACTACCTCATGCGTCTATT 7a_seq TAGTGGAGCCCGCTATCTTCTTTAACTATACA ACCTA 59 let- AAAATGTCGTTGCCCTCCTACCTCATGCGTCT 7e_seq ATTTAGTGGAGCCGCCTATCTTCTTTAACTAT ACAAC 60 let- AAAATGTCGTTGCCCTACTACCTCTTGCGTCT 7d_seq ATTTAGTGGAGCCATCTATCTTCTTTAACTAT GCAAC 61 let- AAAATGTCGTTGCTCTACTACCTCATGCGTCT 7f_seq ATTTAGTGGAGCCTACTATCTTCTTTAACTAT ACAAT 62 Decor- Cy3-TGCGTCTATTTAGTGGAGCCa ator probe 63 anchor AlexaFluor750-TGCGTCTATTTAGTGGA primer GCCa 64 seglibb pTNNNNNNNN-FITCb 1T 65 seqlibb PGNNNNNNNN-Cy3b 1G 66 seglibb pANNNNNNNN-Cy5b 1A 67 seqlibb PCNNNNNNNN-TexasRedb 1C
TABLE-US-00004 TABLE 4 SEQ ID NO: Name Sequence (5′-3′) 68 Mouse GGCCTGTACACTGACTTGAGACCAATAAAAGTGCA ACTB CACCTTACCTTACACAAAC 69 Human TACCTGTACACTGACTTGAGACCAGTTGAATAAAA ACTB GTGCACACCTTAAAAATGAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA 70 Mouse TAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTA PLP AAAGTAGCCGTGACTATCGACT GTTTGTGTAAGG 71 Human TAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAA PLP GTCGGAAGTACTACTCTCTTTTTTTTTCATTTT 72 Mouse TAAGGTGTGCAAAAAGTAGCCGTGACTATCGACTA CPLP AAAGTAGCCGTGACTATCGACTGTTTGTGTAAGrG 73 Human TAAGGTGTGCAAGTCGGAAGTACTACTCTCTAAAA cPLP GTCGGAAGTACTACTCTCTTTTTTTTTCATTTrU 74 Mouse TAtaTCcctatatGTAAGGTGTGCAAAAAGTAGCC iLock GTGACTATCGACTAAAAGTAGCCGTGACTATCGAC TGTTTGTGTAAGG 75 Human TAtaTCcctatatTTAAGGTGTGCAAGTCGGAAGT iLock ACTACTCTCTAAAAGTCGGAAGTACTACTCTCTTT TTTTTTCATTTT 76 Mouse TAtaTCcctatatrGTAAGGTGTGCAAAAAGTAGC ciLock CGTGACTATCGACTAAAAGTAGCCGTGACTATCGA CTGTTTGTGTAAGrG 77 Human TAtaTCcctatatrUTAAGGTGTGCAAGTCGGAAG ciLock TACTACTCTCTAAAAGTCGGAAGTACTACTCTCTT TTTTTTTCATTTrU
TABLE-US-00005 TABLE 5 78 miR21_BIO rUrUrUrUrUrUrUrUrUrUrUrUrUrUrU rUrArGrCrUrUrArUrCrArGrArCrUrG rArUrGrUrUrGrA 79 >miR21B2DO_ /5Phos/CTGATAAGCTAGCCGAATCTAAG PLP AGTAGCCGTGACTATCGACTAAAACTACAC CA 80 >miR21B2DO_ ccgtcgctgcgtTCTGATAAGCTAAAAAAA iLock AGTAGCCGTGACTATCGACTAAAAAAATCA ACATCAGT 81 > miR21 /5Phos/CTGATAAGCTAGCCGAATCTAAG B2DO_rPLP AGTAGCCGTGACTATCGACTAAAACTACAC CATCAACATCAGrU 82 >miR21B2DO_ ccgtcgctgcgtrUCTGATAAGCTAAAAAA RiLock AAGTAGCCGTGACTATCGACTAAAAAAATC AACATCAGrU 83 >B2DO_Cy3 /Cy_3/AGTAGCCGTGACTATCGACT 84 >antimiR21_ FAM/TCAACATCAGTCTGATAAGCTA FAM 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 ACATTTTCATTATTTTTATTATAAGGCCTGCT template GAAAATGACTGAATATAAACTTGTGGTAGTTG GAGCTGGTGGCGTAGGCAAGAGTGCCTTGACG ATA 86 wt KRAS AGCTCCAACTACCACAAAGTCGATAGTCACGG PLP CTACTCAGACGTAACGCGTTCAGTGATGCCCT TGCCTACGCCACC 87 KRAS RNA rArArCrUrUrGrUrGrGrUrArGrUrUrGrG template rArGrCrUrGrGrUrGrGrCrGrUrArGrGrC rArArGrArGrUrGrCrC 88 chim wt AGCTCCAACTACCACAAAGTCGATAGTCACGG KRAS PLP CTACTCAGACGTAACGCGTTCAGTGATGCCCT TGCCTACGCCACrC 89 chim wt AGCTCCAACTACCACAAAGTCGATAGTCACGG KRAS CTACTCAGACGTAACGCGTTCAGTGATGCCCT PLP_2 TGCCTACGCCArCrC 90 mut KRAS AGCTCCAACTACCACAACCTCAATGCACATGT PLP TTGGCTCCCAGACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACT 91 chim mut AGCTCCAACTACCACAACCTCAATGCACATGT KRAS PLP TTGGCTCCCAGACGTAACGCGTTCAGTGATGC CCTTGCCTACGCCACrU 92 RCA primer CAT CAC TGA ACG C*G*T 93 mut DO-Cy5 CCTCAATGCACATGTTTGGCTCC 94 wt DO-Cy3 AGTCGATAGTCACGGCTACT 95 KRAS wt TAtaTCcctatatrCAGCTCCAACTACCACAA invader AGGTCGATAGTCACGGCTACTCAGACGTAACC chim 3-5 GTTCAGTGATGCCCTTGCCTACGCCACrC 96 KRAS wt TAtaTCcctatatCAGCTCCAACTACCACAAA invader GTCGATAGTCACGGCTACTCAGACGTAACGCG chim 3 TTCAGTGATGCCCTTGCCTACGCCACrC 97 KRAS mut TAtaTCcctatatrUAGCTCCAACTACCACAA invader CCTCAATGCACATGTTTGGCTCC CAGACGT chim 3-5 AACGCGTTCAGTGATGCCCTTGCCTACGCCA CrU 98 KRAS mut TAtaTCcctatatTAGCTCCAACTACCACAAC invader CTCAATGCACATGTTTGGCTCC CAGACGTA chim 3 ACGCGTTCAGTGATGCCCTTGCCTACGCCA CrU
TABLE-US-00007 TABLE 7 99 KRAS gapfill TAtaTCtctatatAGCTCCAACTACCACA invader AGTAGTCGATAGTCACGGCTACTCAGACG TAACGCGTTCAGTGATGAGTAGGC ACTCTTGCCTAC 100 invader_ TAtaTCtctatatGCCACrC splint_chim 101 invader_ TAtaTCtctatat GCCACC splint 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 5′ ID modifi- NO Name cation Sequence (5′-3′) 106 Kras_ rArArCrUrUrGrUrGrGrUrArG wt_RNA rUrUrGrGrArGrCrUrGrGrUrG rGrCrGrUrArGrGrCrArArGrA rGrUrGrCrC 107 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_1 CArGACGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 108 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_2 CArGrACGTAACGrCrGTTCAG TGATGCCCTTGCCTACGCCACC 109 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_3 CArGrArCGTAACGrCrGTTCA GTGATGCCCTTGCCTACGCCACC 110 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_4 CArGrArCrGTAACGrCrGTTCA GTGATGCCCTTGCCTACGCCACC 111 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_5 CArGrArCrGrUAACGrCrGTTC AGTGATGCCCTTGCCTACGCCACC 112 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_7 CArGrArCrGrUrArACGrCrG TTCAGTGATGCCCTTGCCTACG CCACC 113 R_KRAS Phos AGCTCCAACTACCACAA [1] wt1_6S CArGrACGrUrAACrGrCGTTC AGTGATGCCCTTGCCTACGCCA CC 114 R_KRAS Phos AGCTCCAACTACCAC (10A) wt1_DNA [2] (10A) CTTGCCTACGCC ACC 115 R_KRAS Phos AGCTCCAACTACCAC (10A) wt1_RNA [2] (10A) CTTGCCTACGC CACrC 116 Decorator Cy3 CCTCAATGCACATGTTTGGCTCª probe [1] 117 Decorator Cy3 AGTCGATAGTCACGGCTACTª probe2 [2] r[N]: RNA oligonucleotide; (10A): linker; 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 rArArCrUrUrGrUrGrGrUrArGrUrUrGrGrArGrCrUrGrGrUrGrG rCrGrUrArGrGrCrArArGrArGrUrGrCrC 119 R_KRAS.0 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCAGACGTAACGCG TTCAGTGATGCCCTTGCCTACGCCACC 120 R_KRAS.1G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGACGTAACGC GTTCAGTGATGCCCTTGCCTACGCCACC 121 R_KRAS.1C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCACGTAACGC GTTCAGTGATGCCCTTGCCTACGCCACC 122 R_KRAS.1A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArAACGTAACGC GTTCAGTGATGCCCTTGCCTACGCCACC 123 R_KRAS.1U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUACGTAACGC GTTCAGTGATGCCCTTGCCTACGCCACC 124 R_KRAS.2G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGCGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 125 R_KRAS.2C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCCGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 126 R_KRAS.2A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArACGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 127 R_KRAS.2U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUCGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 128 Alul KRAS CGCCACCAGCTCCAACTA R0 129 PE1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGGTGGCGTAGGCAAG GGC 130 PE2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCAACTACCACAAA GTCG 131 R_KRAS.3G AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrGrGTTAAC GrCrGTTCAGTGATGCCCTTGCCTACGCCACC 132 R_KRAS.3C AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArCrCrCGTAAC GrCrGTTCAGTGATGCCCTTGCCTACGCCACC 133 R_KRAS.3A AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArArArAGTAAC GrCrGTTCAGTGATGCCCTTGCCTACGCCACC 134 R_KRAS.3U AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArUrUrUCGTAA CGrCrGTTCAGTGATGCCCTTGCCTACGCCACC 135 R_KRAS.5 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCrGrUA ACGCGTTCAGTGATGCCCTTGCCTACGCCACC 136 R_KRAS.6S AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrACGrUrAA CrGrCGTTCAGTGATGCCCTTGCCTACGCCACC 137 R_KRAS.3 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrArCGTAAC rGrCrGTTCAGTGATGCCCTTGCCTACGCCACC 138 R_KRAS.3S AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGArCGrUAAC GCGTTCAGTGATGCCCTTGCCTACGCCACC 139 R_KRAS.2 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCArGrACGTAACG rCrGTTCAGTGATGCCCTTGCCTACGCCACC 140 R_KRAS.7 AGCTCCAACTACCACAAAGTCGATAGTCACGGCTACTCA rGrArCrGrUrArACGCGTTCAGTGATGCCCTTGCCTACGCCACC 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: Alul restriction site, these padlock probes were used in the monomer sequencing experiment.
TABLE-US-00010 TABLE 10 141 Detection 5′ Alexa750-TGCGTCTATTTAGTGGAGCC probe (seq anchor probe) 142 Seq library 5′ phos-GNNNCTATC-3′ Cy3 1.sup.st base-G 143 RCA primer 5′-GGCTCCACTAAATAGACG*C*A-3′ thiophosphate (*) 144 Seq library 5′ phos-ANNNCTATC-3′ Cy5 1.sup.st base-A 145 Seq library 5′ phos-CNNNCTATC-3′ Texred 1.sup.st base-C 146 Seq library 5′ phos-TNNNCTATC-3′ Alexa488 1.sup.st base-T 147 Seq library 5′ phos-NGNNCTATC-3′ Cy3 2.sup.nd base-G 148 Seq library 5′ phos-NANNCTATC-3′ Cy5 2.sup.nd base-A 149 Seq library 5′ phos-NCNNCTATC-3′ Texred 2.sup.nd base-C 150 Seq library 5′ phos-NTNNCTATC-3′ Alexa488 2.sup.nd base-T 151 Seq library 5′ phos-NNGNCTATC-3′ Cy3 3.sup.rd base-G 152 Seq library 5′ phos-NNANCTATC-3′ Cy5 3.sup.rd base-A 153 Seq library 5′ phos-NNCNCTATC-3′ Texred 3.sup.rd base-C 154 Seq library 5′ phos-NNTNCTATC-3′ Alexa488 3.sup.rd base-T 155 Seq library 5′ phos-NNNGCTATC-3′ Cy3 4.sup.th base-G 156 Seq library 5′ phos-NNNACTATC-3′ Cy5 4.sup.th base-A 157 Seq library 5′ phos-NNNCCTATC-3′ Texred 4.sup.th base-C 158 Seq library 5′ phos-NNNTCTATC-3′ Alexa488 4.sup.th base-T Chimeric probe name RNA directed chimeric probe sequence 159 Calb_1119_RNA CATCGCAGCGGAGACGACAGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAATGCTATCTTCTTTATACAGCGAAGAACTCATrG 160 Calb2_1328_RNA CACACACGTCAAGAACACAACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAATGCTATCTTCTTTGAAGCCAAAGAGAAAAGGArA 161 Calb2_164_RNA ACCTTCAATGTACCCATTTCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAATGCTATCTTCTTTAAGAAGTTCTCTAGCTCTTrU 162 Calb2_500_RNA AGGTTCATCATAGGGCCTGTCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAATGCTATCTTCTTTTGGGTGTACTCCTGGAGCTrU 163 Calb2_937_RNA GTCAAGAGAGTCAGGACAGCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAATGCTATCTTCTTTGGGGAGGTCTGGGAAGGAGrU 164 Calb2_DO3_CCAA_ CTCATACAGATCCTTCAGCTGATTCCTTTGACTCACATTGCGTCTATTT RNA AGTGGAGCCCCAACTATCTTCTTTTTCATCTCCTTCTTGTTCTrU 165 Chod1_1164_RNA CGGGCTAGTTTTTGATCTTCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTATCCACAGTGTAGACTGATrU 166 Chod1_1798_RNA AAAGCAAAGAAACAGAACAACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTTCCTAAACTTTATCGAACCrC 167 Chod1_2071_RNA ATTCTATAGGCAACATGTGACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTACTCTGGGGAGCTATTTGCrA 168 Chod1_2252_RNA GTTCTGCTTAGCATCACACTCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTTTAATCATTAATATCAGTGrU 169 Chod1_293_RNA TCCTACTCCCTCCTTCCCAGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTTTCCTTGCTTTCCTGCTGGrG 170 Chod1_789_RNA GAACTGGGAGCTGCTTCCATCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTTCATCAGTGTACCAGTTTCrG 171 Chod1_916_RNA TGTTGCACCTGTCGTCATTCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAGTACTATCTTCTTTGCAGATGTAATTGTGCTTCrA 172 Cort_326_RNA CCCGGGGGTACCCCCTCCGACGTGCTTGTGGTAGCAAATATGCGTCTAT TTAGTGGAGCCTGATCTATCTTCTTTCTTTCCTGGCTCTTGGACArG 173 Cort_529_RNA CAAAGCTGACATAAGAAGAACGTGCTTGTGGTAGCAAATATGCGTCTAT TTAGTGGAGCCTGATCTATCTTCTTTTTCTCACAGGGCAGGGGAGrG 174 Htr3a_1014_ AGTGTGTCTGACACGATGATCGTGCTTGTGGTAGCAAATATGCGTCTAT GGAC_RNA TTAGTGGAGCCGGACCTATCTTCTTTTACCGATGGCCGTTGCTGGrC 175 Htr3a_1309_ ATGGCTGCAGTGGTTTCCCACGTGCTTGTGGTAGCAAATATGCGTCTAT GTAA_RNA TTAGTGGAGCCGTAACTATCTTCTTTAAGTCCTGAGGTCCTCCAArC 176 Htr3a_1573_ CCAAATGGACCAGAGAGTGACGTGCTTGTGGTAGCAAATATGCGTCTAT TAAC_RNA TTAGTGGAGCCTAACCTATCTTCTTTGTGCCCACTCAAGAATAATrG 177 Htr3a_1750_ AAGTCAGAGAGACAGACTGGCGTGCTTGTGGTAGCAAATATGCGTCTAT TATT_RNA TTAGTGGAGCCTATTCTATCTTCTTTGCTTTAAAGCCATGATAGGrG 178 Htr3a_1927_ GCAAGACAATTTGCTTTTCTCGTGCTTGTGGTAGCAAATATGCGTCTAT TCGC_RNA TTAGTGGAGCCTCGCCTATCTTCTTTCAGAAGTCTCAGGCATCTArU 179 Htr3a_2045_ ATTATCCCCTGCTCCCATTGCGTGCTTGTGGTAGCAAATATGCGTCTAT TGGA_RNA TTAGTGGAGCCTGGACTATCTTCTTTTTAAGATATCATAGCATTTrU 180 Htr3a_21_ GTCCCAGGCAGACTGCTTTTCGTGCTTGTGGTAGCAAATATGCGTCTAT AAGT_RNA TTAGTGGAGCCAAGTCTATCTTCTTTCCACCCGCTGCCAACCTCArU 181 Htr3a_247_ GTCTGACAGCCTTAGTAGAGCGTGCTTGTGGTAGCAAATATGCGTCTAT CAGC_RNA TTAGTGGAGCCCAGCCTATCTTCTTTTTGTAGTTAGCCAGGAGGTrG 182 Htr3a_424_ AGTCCACTGCAGAAACTCATCGTGCTTGTGGTAGCAAATATGCGTCTAT CGGT_RNA TTAGTGGAGCCCGGTCTATCTTCTTTACATTGTCGAAGTCCTCAGrG 183 Htr3a_89_ CTCAGAGCAGCCACTCAGGACGTGCTTGTGGTAGCAAATATGCGTCTAT CACT_RNA TTAGTGGAGCCCACTCTATCTTCTTTCTTCCCAGATGTGGGAGGGrC 184 Htr3a_955_ AGAGACTCTCTCACCGCTGTCGTGCTTGTGGTAGCAAATATGCGTCTAT GCTC_RNA TTAGTGGAGCCGCTCCTATCTTCTTTAGAAGGAGTGTGATCTTGArA 185 Neurod6_ TAGAAGGATTCATATGCACTCTGATTCCTTTGACTCACATTGCGTCTAT 1033_RNA TTAGTGGAGCCTTGCCTATCTTCTTTACTCAGGGGAGGTACTTTCrA 186 Neurod6_ CTGCTAGTGACGTCACAGGGCTGATTCCTTTGACTCACATTGCGTCTAT 108_RNA TTAGTGGAGCCTTGCCTATCTTCTTTAGAGCTGGTACCCATGCCArU 187 Neurod6_ TGTGATACAGACAAGAGGGACTGATTCCTTTGACTCACATTGCGTCTAT 1524_RNA TTAGTGGAGCCTTGCCTATCTTCTTTAGAGAGAGAGAGAATCACArG 188 Neurod6_ TCTCATTGATCTCTAAAAAGCTGATTCCTTTGACTCACATTGCGTCTAT 168_RNA TTAGTGGAGCCTTGCCTATCTTCTTTATCTGTGTGTATCTGCACTrA 189 Neurod6_ AGACATTGAAGTATGCTGTGCTGATTCCTTTGACTCACATTGCGTCTAT 1688_RNA TTAGTGGAGCCTTGCCTATCTTCTTTCATAACTGTACAACTGAAArU 190 Neurod6_ AACAATACAAAACAAGTGCTCTGATTCCTTTGACTCACATTGCGTCTAT 2041_RNA TTAGTGGAGCCTTGCCTATCTTCTTTACCTGTACAGAAAAATCCTrG 191 Neurod6_ TTTTCAGGCTGAGTGTCGCACTGATTCCTTTGACTCACATTGCGTCTAT 228_RNA TTAGTGGAGCCTTGCCTATCTTCTTTCATTTTGGATCTTCCAAATrC 192 Neurod6_ GTAGTGTTAACATGGTTCTTCTGATTCCTTTGACTCACATTGCGTCTAT 315_RNA TTAGTGGAGCCTTGCCTATCTTCTTTTACGACAGACTCGTCAAACrG 193 Neurod6_ TGTCTTCTTCCTCCTCTTCTCTGATTCCTTTGACTCACATTGCGTCTAT 495_RNA TTAGTGGAGCCTTGCCTATCTTCTTTATTCTCATCTTCTTCCTCTrC 194 Neurod6_ TGTCCAGAGCATCATTGAGGCTGATTCCTTTGACTCACATTGCGTCTAT 648_RNA TTAGTGGAGCCTTGCCTATCTTCTTTGGGGACCACTTTTCGCAAArU 195 Neurod6_ GTCGTAAAGTTTCTATTTTGCTGATTCCTTTGACTCACATTGCGTCTAT 714_RNA TTAGTGGAGCCTTGCCTATCTTCTTTCCAGATGTAATTTTTGGCCrA 196 Neurod6_ CCCATGCCCTGGGGGAGTGGCTGATTCCTTTGACTCACATTGCGTCTAT 971_RNA TTAGTGGAGCCTTGCCTATCTTCTTTGACTTGGAATTATCAAGAGrU 197 Nov_DO2_ GGAGAAAGTTCATGACACTCTACGATTTTACCAGTGGCTGCGTCTATTT ATGC_RNA AGTGGAGCCATGCCTATCTTCTTTGAGTCGGTTTGTCTATAArU 198 Pcp4_120_RNA TCAGAAGGCAATGCTCAGGGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCATCCCTATCTTCTTTGCTAGGTCCCACAGAACAGrC 199 Pcp4_181_RNA TCCGGCACTTTGTCTCTCACCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCATCCCTATCTTCTTTTTGTCTTTTCCGTTGGTCGrC 200 Pcp4_305_RNA ACTGAGACTGAATGGCCACACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCATCCCTATCTTCTTTTTTCTTCTGGAATTTTCTGrA 201 Pcp4_386_RNA AACTTGGTGTCTTCAGGTGGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCATCCCTATCTTCTTTTTCTTGATGGATGGTGGTTrG 202 Pcp4_472_RNA TTCAGGTTTGTAGCAGGGTGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCATCCCTATCTTCTTTATGGGTTTCTCTTCATGCArU 203 Pdela_1081_RNA TTCTCTGTTGAGTCCGTCAGTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTCTATAAGAGGAATGACAATrU 204 Pdela_120_RNA ACTTTGGTTTTTCTTCAGGCTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTAGCATGGACAATGCTGCGArA 205 Pdela_1216_RNA TCACACACGGAGCCTTTTGTTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTAGTCTGGGGCATAGCTCCCrA 206 Pdela_273_RNA CATTTAAGGCAAATACATCATCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTGCTATGCTCCCCGCTTGCTrU 207 Pdela_334_RNA AGATCATATCTGGTAAAGAGTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTGAATCTTGAAGCGGTTGATrA 208 Pdela_469_RNA ATATAATGCACAGTTTGAGTTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTTGATACCTGTATGAAGCATrU 209 Pdela_615_RNA TGTACAGAATAGCAACGTCCTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTCTCAAGCACTGAGCGGTCGrU 210 Pdela_759_RNA CTGTCGCTAAGACCATTTCATCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTCTGAAAATGCCCTGACATGrU 211 Pdela_904_RNA CTGTAGTGCAACTTCCAAGTTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTCCATTAGGGCCATGGTCCArU 212 Pdela_995_RNA CTTCCGATCACAAAGTGGAGTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCATCGCTATCTTCTTTGACTGGGCAACCATTGTTGrA 213 Penk_1282_RNA CAATACTGAGCTTCAAGACTTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCGTACCTATCTTCTTTACAACATAGCCATAAGAGArC 214 Penk_ 286_RNA CATGGGCTGTAGGAGAGAAGTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCGTACCTATCTTCTTTCAAAGCCTCAGGAACCGCGrC 215 Penk_638_RNA GATATAGCTCGTCCATCTTCTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCGTACCTATCTTCTTTTTCTTCTTCTGGCTCCATGrG 216 Penk_83_RNA TGCCTGGGACTATTCTATCTTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCGTACCTATCTTCTTTGTTTCCTGCTGTTCTAGTGrA 217 Penk_882_RNA AGTTGGGGGCTTCTTTTGAGTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCGTACCTATCTTCTTTGCTCTTTTGCTTCATCTTCrC 218 Plcxd2_DO3_ GCACTCCTACACAATGACTGATTCCTTTGACTCACATTGCGTCTATTTA GAAA_RNA GTGGAGCCGAAACTATCTTCTTTGAAGATGGTGAGGGTArU 219 Plcxd2_DO3_ GTGGTAGAAAATGAGAACCTGATTCCTTTGACTCACATTGCGTCTATTT GGCC_RNA AGTGGAGCCGGCCCTATCTTCTTTTGCTTGTAGAAGGGACrA 220 Rorb_2282_RNA TCATTCAGAATTGGATTCCACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAAATCTATCTTCTTTATAACCACCAAAGTGAAGTrU 221 Rorb_4479_RNA TCAATTTTCTGCCTTAAGCCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAAATCTATCTTCTTTAAGAAGAAAAAGAAGTTCArU 222 Rorb_536_RNA ATGTGAGGTCATAGATAGGTCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAAATCTATCTTCTTTGGTAAACAAGTTGGGTACArG 223 Rorb_6395_RNA GAGAAAGTGTCACAGATTTGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAAATCTATCTTCTTTAGGTACAATTAAGAGAAAGrG 224 Rorb_8435_RNA TAGTTGTTAGGGAGTGCTGCCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCAAATCTATCTTCTTTAAGTAATAGAAAACTCTTTrU 225 Rorb_DO3_ AGCCTTTTAAAGTCATATTTGGTCTGATTCCTTTGACTCACATTGCGTC CCGG_RNA TATTTAGTGGAGCCCCGGCTATCTTCTTAATCGGTCATCATAAAATACrU 226 Rprm_654_RNA CGGTCCGTGATGGTGCGTGCTTGTGGTAGCAAATATGCGTCTATTTAGT GGAGCCTTGTCTATCTTCTTTGACAGGTTTGCGTTGCrU 227 Sst_432_ TAACAGGATGTGAATGTCTTCTACGATTTTACCAGTGGCCTGACTATCT AP1SWPLP_RNA TCTTTTAGGACAACAATATTAAAGrC 228 Synpr_1071_RNA CATACTAGAGACTTTAAGCTICTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCATTACTATCTTCTTTAAGGTAATCTATGCACATTrA 229 Synpr_1643_RNA CCTCTCTGGATGCAAAGAAATCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCATTACTATCTTCTTTAACTATGGTGTCTAAATCTrG 230 Synpr_260_RNA ATGTACACGACCGTGGCAGCTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCATTACTATCTTCTTTGGTACTTGTTCTGGAAGAArA 231 Synpr_591_RNA AAGGTATCTCTGTCCAGAGGTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCATTACTATCTTCTTTTGCTTCTCCATGGGGTCTGrA 232 Synpr_DO2_ CTTAAAAATTCTTCTGCTACTGGTCTACGATTTTACCAGTGGCTGCGTC ATTA_RNA TATTTAGTGGAGCCATTACTATCTTCTTCATTAATAATTGATTGAAACrU 233 Yjefn3_138_RNA CACTAGCGTGCCCACATAGTCGTGCTTGTGGTAGCAAATATGCGTCTAT TTAGTGGAGCCTTTACTATCTTCTTTGGCCTTGGTCACAGCCACCrG 234 Yjefn3_344_RNA CTTCTCGCACTGCGTGGTCACGTGCTTGTGGTAGCAAATATGCGTCTAT TTAGTGGAGCCTTTACTATCTTCTTTGACAGGAAGGGGATGTCCArU 235 Yjefn3_686_RNA AAACTTTCGGCGGACGTCATCGTGCTTGTGGTAGCAAATATGCGTCTAT TTAGTGGAGCCTTTACTATCTTCTTTTATTTTGGCAGGTGCAGGCrC 236 Bgn_412_RNA GGGCACAGTCTTCAGACCCATCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCCTATCTATCTTCTTTGTGTCAGGTGAGATCTCCTrU 237 Bgn_851_RNA TCAGGGTCTCAGGGAGATCTTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCCTATCTATCTTCTTTGTGGTCCAGGTGAAGTTCGrU 238 Bgn_1194_RNA TCCCAGTAGGGCACAGGGTTTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCCTATCTATCTTCTTTGGAAGGTGGCAGGCTGCACrU 239 Bgn_1577_RNA AACAATGGCGGTGGCAGTGTTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCCTATCTATCTTCTTTAGGAACACATGCCTGGATGrG 240 Bgn_2309_RNA TCAGGGACCCAGGGGTGAGGTCTACGATTTTACCAGTGGCTGCGTCTAT TTAGTGGAGCCCTATCTATCTTCTTTGACCATCACCTCCTACCACrA 241 Apq4_879_RNA CTTAAGGCGACGTTTGAGCTCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCCATTCTATCTTCTTTGCGGCTTTGCTGAAGGCTTrC 242 Apq4_2186_RNA AATTACACTCACAATGCCGACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCCATTCTATCTTCTTTTAATTCACACAAATGGGTArU 243 Apq4_3100_RNA CACTGGAAATGACTGTTAAACTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCCATTCTATCTTCTTTTGTACCATACTGAATGCTGrU 244 Apq4_3673_RNA CGGTGTATCTGTCAGTAGCTCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCCATTCTATCTTCTTTTTTCCTCTCTGATCTCTGTrG 245 Apq4_4344_RNA ACAGAGGCAGTGTCTCTGTGCTGATTCCTTTGACTCACATTGCGTCTAT TTAGTGGAGCCCATTCTATCTTCTTTGCTCTCTGGCTTCAATTGTrC 246 Pdgfra_296_RNA TGGGAGGATAGAGGGTAATATCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCTGTCCTATCTTCTTTACAATCTTCTCATTCTCGTrU 247 Pdgfra_646_RNA GGTATGATGGCAGAGTCATCTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCTGTCCTATCTTCTTTCCGGATCTGTGGTGCGGCArA 248 Pdgfra_832_RNA GTTGCTTTCAAGGCATAAACTCTACGAGTTTGCAGTCACGTGCGTCTAT TTAGTGGAGCCTGTCCTATCTTCTTTTCTCCAGATTCAGTTCTGArC 249 Pdgfra_1227_ GGTAGGCCTGCACCTCCACCTCTACGAGTTTGCAGTCACGTGCGTCTAT RNA TTAGTGGAGCCTGTCCTATCTTCTTTCCACGATATCCTGGGCGTCrG 250 Pdgfra_1544_ CATCCAGTCGATTTCTGGAATCTACGAGTTTGCAGTCACGTGCGTCTAT RNA TTAGTGGAGCCTGTCCTATCTTCTTTTTCTTAATATGCTTGCAGArU
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 AATGATACGGCGACCACCGAGATCTACAC AGGCTATAACACTCTTTCCCTACACGAC 252 fwd_index2 AATGATACGGCGACCACCGAGATCTACAC GCCTCTATACACTCTTTCCCTACACGAC 253 fwd_index3 AATGATACGGCGACCACCGAGATCTACAC AGGATAGGACACTCTTTCCCTACACGAC 254 fwd_index4 AATGATACGGCGACCACCGAGATCTACAC TCAGAGCCACACTCTTTCCCTACACGAC 255 fwd_index5 AATGATACGGCGACCACCGAGATCTACAC CTTCGCCTACACTCTTTCCCTACACGAC 256 fwd_index6 AATGATACGGCGACCACCGAGATCTACAC TAAGATTAACACTCTTTCCCTACACGAC 257 fwd_index7 AATGATACGGCGACCACCGAGATCTACAC ACGTCCTGACACTCTTTCCCTACACGAC 258 fwd_index8 AATGATACGGCGACCACCGAGATCTACAC GTCAGTACACACTCTTTCCCTACACGAC 259 rev_index1 CAAGCAGAAGACGGCATACGAGAT CGAGTAATGTGACTGGAGTTCAGACGTGT 260 rev_index2 CAAGCAGAAGACGGCATACGAGAT TCTCCGGAGTGACTGGAGTTCAGACGTGT 261 rev_index3 CAAGCAGAAGACGGCATACGAGAT AATGAGCGGTGACTGGAGTTCAGACGTGT 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_KRA AGCTCCAACTACCACAAAGTCGArUAGTCACGGCTACT S.D3 CArGACGTAACGCGTTCAGrUGATGCCCTTGCCTACGC CACC 265 R_KRA AGCTCCAACTACCACAArAGTCGArUAGTCArCGGCTA S.D8 rCTCAGArCGTAACGrCGTTCAGrUGATGCrCCTTGCC TACGCCACC
TABLE-US-00013 TABLE 13 267 R_KRAS.2G AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArGrGCGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 268 R_KRAS.2C AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArCrCCGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 269 R_KRAS.2A AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArArACGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 270 R_KRAS.2U AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArUrUCGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 271 R_KRAS.3G AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArGrGrGTTAACGCGTTCAGTG ATGCCCTTGCCTACGCCACC 272 R_KRAS.3C AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArCrCrCGTAACGCGTTCAGTG ATGCCCTTGCCTACGCCACC 273 R_KRAS.3A AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArArArAGTAACGCGTTCAGTG ATGCCCTTGCCTACGCCACC 274 R_KRAS.3U AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArUrUrUCGTAACGCGTTCAGT GATGCCCTTGCCTACGCCACC 275 R_KRAS.3 AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArGrArCGTAACGCGTTCAGTG ATGCCCTTGCCTACGCCACC 276 R_KRAS.2 AGCTCCAACTACCACAAAGTCGATAGTCAC GGCTACTCArGrACGTAACGCGTTCAGTGA TGCCCTTGCCTACGCCACC 277 R_KRAS AGCTCCAACTACCACAA [1] wt1_2 CArGrACGTAACGCGTTCAGTGATGCCCTT GCCTACGCCACC 278 R_KRAS AGCTCCAACTACCACAA [1] wt1_3 CArGrArCGTAACGCGTTCAGTGATGCCCT TGCCTACGCCACC 279 R_KRAS AGCTCCAACTACCACAA [1] wt1_4 CArGrArCrGTAACGCGTTCAGTGATGCCC TTGCCTACGCCACC 280 R_KRAS AGCTCCAACTACCACAA [1] wt1_5 CArGrArCrGrUAACGCGTTCAGTGATGCC CTTGCCTACGCCACC 281 R_KRAS AGCTCCAACTACCACAA [1] wt1_7 CArGrArCrGrUrArACGCGTTCAGTGATG CCCTTGCCTACGCCACC
TABLE-US-00014 SEQ ID NO: 266: Bacteriophage Phi29 WT polymerase MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAW VLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQW YMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHK ERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKD IITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDV NSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIP TIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISG LKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVT GKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYD RIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTY IQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGF SRKMKPKPVQVPGGVVLVDDTFTIK