Method for in-situ analysis of single nucleotide variation in RNA sample and kit for in-situ analysis of single nucleotide variation in RNA sample

Abstract

The present disclosure relates to a method for in situ analysis of single nucleotide variations in an RNA sample, the method comprising the steps of: (a) contacting the RNA sample with a plurality of chimeric padlock probes comprising a modified base at the 3'end portion; (b) adding an RNA ligase to generate a cyclized padlock oligonucleotide for a padlock probe comprising a modified base complementary to the corresponding position of the RNA sample under conditions and reagents allowing the ligation of the padlock probe with single nucleotide specificity; (c) amplifying the cyclized padlock oligonucleotide under conditions and reagents that allow rolling circle amplification, thereby producing an amplified cyclized padlock oligonucleotide; and (d) detecting the amplified cyclized padlock oligonucleotide so as to obtain a map of the mononucleotide in the RNA sample. The disclosure further relates to a kit for in situ analysis of single nucleotide variations in an RNA sample.

Description

Technical Field

[0001] The present disclosure relates to a method for in situ analysis of single nucleotide variations in RNA samples and a kit for use in in situ analysis of single nucleotide variations in RNA samples. Background Art

[0002] The use of RNA-templated padlock probe ligation followed by rolling circle amplification to detect mRNA is a recently established method for in situ mRNA analysis. Currently, this method is based on two alternative but similar methodological variants: 1) ligation of DNA padlock probes to target mRNA using SplintR ligase ( https: / / doi.org / 10.1371 / journal.pbio.3000675 ), or 2) using T4 RNA ligase 2 (T4 Rnl2) to ligate chimeric RNA probes (DNA probes with a 3'-terminal ribose) to the target mRNA (doi:10.1261 / rna.066753.118). However, both ligases exhibit high mismatch tolerance (doi:10.1261 / rna.066753.118). This presents two major obstacles: On the one hand, padlock probes cannot be used to directly distinguish between SNPs or even small deletions on the target mRNA. On the other hand, RNA-templated padlock probe ligation cannot distinguish between RNAs of closely related genes (i.e., recent gene duplications whose sequences have not yet diverged significantly).

[0003] Krzywkowski et al. (RNA, 2019 Jan;25(1):82-89, “Chimeric padlock and iLockprobes for increased efficiency of targeted RNA detection”) investigated the use of padlock technology with DNA ligase on RNA substrates. However, RNA detection sensitivity was found to be limited.

[0004] Therefore, the current state-of-the-art method for detecting single nucleotide variants on mRNA requires first performing reverse transcription to generate cDNA from mRNA in situ, and then using DNA padlock probes with a very accurate DNA ligase (Tth) to detect single nucleotide variants on the generated cDNA. However, the efficiency of in situ cDNA synthesis is low and will cause a detection bottleneck, which leads to low detection efficiency of single nucleotide variant events ( https: / / dx.doi.org / 10.18632%2Foncotarget.1527 ).

[0005] Therefore, there are currently no appropriate methods and / or reliable approaches to directly analyze single nucleotide polymorphisms or variations (SNPs / SNVs) on mRNA in biological tissues in situ. The low detection efficiency of cDNA methods generally fails to produce high-quality data, especially when detecting mRNAs with lower abundance. In addition, known cDNA-based methods for SNP detection perform poorly in FFPE tissue sections, possibly due to fragmented mRNA content. The most advanced method for efficient SNP analysis today is to use single-cell RNA sequencing (scRNA-seq) methods. Although the scRNA-seq method is efficient, it requires the separation of biological tissues during the process, resulting in the loss of spatial information.

[0006] Spatial information is crucial because biology is inherently heterogeneous, and without spatial information about cells, researchers cannot infer cell-to-cell interactions within the tissue itself. This type of SNP spatial information is particularly important in immuno-oncology research, where researchers hope to gain a deeper understanding of diseases such as cancer.

[0007] cDNA-based in situ sequencing (ISS) methods seek to address the spatial aspects of in situ SNP analysis, but due to inefficiencies in cDNA synthesis from mRNA and post-fixation, this approach suffers from low detection efficiency and is challenging to process in FFPE sections where RNA fragmentation is known (Gyllborg et al., “Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue,” Nucleic Acids Research, vol. 48, no. 19, 4 November 2020, p. e112, https: / / doi.org / 10.1093 / nar / gkaa792 ). WO2022087273 discloses methods for spatial analysis using padlock probe technology followed by rolling circle amplification to facilitate the identification of alternative splicing events, translocation events, and mutations that alter the hybridization rate of one or two probe oligonucleotides. Template-dependent ligation of oligonucleotides that hybridize to a proximal target region of an analyte is discussed in several examples, including the recently characterized archaeal RNA ligase (KOD ligase) from Thermoccocus kodakarensis.

[0008] In addition, WO2019068880 discloses a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample. The use of various enzymes is discussed, including SplintR ligase and T4 RNA ligase II as ligases, and Phi29 DNA polymerase as a polymerase. In addition, the use of a padlock probe with a flap at the 5' end of the probe is also disclosed. In addition, WO2022256422 discloses an asymmetric padlock probe (PLP) for sample SNP and point mutation analysis.

[0009] Therefore, the disclosures of the prior art neither address the problem of analyzing and identifying variations and mutations in RNA molecules in a reliable and efficient manner, nor provide a sufficiently effective solution. Therefore, there is a need for improved methods and approaches to enable analysis of variations in RNA molecules, particularly in in situ applications. Summary of the Invention

[0010] The purpose of the present disclosure is to mitigate, alleviate or eliminate one or more of the above-mentioned defects and shortcomings in the prior art and to solve at least one of the above-mentioned problems.

[0011] The present inventors have developed a method using chimeric padlock probes and single nucleotide-specific ligases, which can directly detect SNPs on mRNA and retain single nucleotide-specific detection, solving the spatial resolution problem, and also providing increased detection sensitivity. Therefore, since the padlock probes (PLPs) can better target fragmented mRNA molecules compared to cDNA methods, this method is suitable for difficult-to-process FFPE tissue sections.

[0012] Therefore, according to a first aspect, there is provided a method for in situ analysis of single nucleotide variations in an RNA sample, the method comprising the following steps:

[0013] (a) contacting the RNA sample with a plurality of chimeric padlock probes under conditions and reagents that allow hybridization, wherein the chimeric padlock probes comprise a modified base at the 3' end portion, wherein the modified base is selected from the group consisting of C, A, G, and U / T;

[0014] (b) adding RNA ligase under conditions and reagents that allow ligation of the padlock probe with single nucleotide specificity to generate a circularized padlock oligonucleotide for the padlock probe comprising a modified base complementary to a corresponding position of the RNA sample;

[0015] (c) amplifying the circularized padlock oligonucleotide under conditions and reagents that allow rolling circle amplification, thereby generating an amplified circularized padlock oligonucleotide;

[0016] (d) detecting the amplified circularized padlock oligonucleotides to obtain a profile of single nucleotides in the RNA sample.

[0017] Therefore, the present invention uses chimeric padlock probes with appropriately modified bases on the 3' end and a SNP-accurate ligase that can ligate the padlock probes with single nucleotide specificity, followed by rolling circle amplification, to analyze single nucleotide variations (SNVs) in situ.

[0018] Whether an RNA ligase exhibits "single nucleotide specificity" can be determined and / or measured by comparing the specificity to a threshold and / or reference value. For example, the threshold and / or reference value can be a comparison of a specific signal and a non-specific signal, wherein, for example, the specific signal can be an order of magnitude higher than the non-specific signal to achieve sufficient specificity and provide a sufficiently strong signal. Alternatively, currently "state-of-the-art" in situ analysis is performed via cDNA methods, which can be used as a benchmark (reference) value.

[0019] Therefore, the inventors have developed a method for directly detecting single nucleotide polymorphisms (SNPs) on mRNA using chimeric padlock probes and ligases. This enables single nucleotide detection in a spatially resolved manner, down to subcellular resolution, while also providing higher detection sensitivity. Furthermore, since padlock probes (PLPs) are better able to target fragmented mRNA molecules compared to cDNA methods, they are suitable for difficult-to-process FFPE tissue sections. Therefore, by cleverly choosing the ligase and padlock probe design, specificity can be achieved that can distinguish and / or analyze single nucleotide variants.

[0020] According to some embodiments, the RNA sample comprises at least one nucleotide position to be analyzed, which is flanked on the 3' and 5' sides by nucleotide fragments with known identities.

[0021] According to some embodiments, the RNA sample is an mRNA sample, an rRNA sample, or a microRNA, or a non-coding RNA.

[0022] According to some embodiments, a plurality of chimeric padlock probes each have a first end and a second end, which are designed to hybridize to nucleotide fragments with known identities at the 3' side and 5' side of the RNA sample and at least one nucleotide position to be analyzed, respectively, so that the terminal base at the 3' end or any one of the two bases next to the terminal base at the 3' end is positioned at the nucleotide position to be analyzed in the RNA sample.

[0023] "Terminal base at the 3' end" generally means the last base at the 3' end.

[0024] According to some embodiments, a padlock probe having a base at its 3' end that is complementary to a nucleotide in a nucleotide position to be analyzed of the RNA sample is ligated in step (c), and wherein a padlock probe having at least one base at its 3' end that is not complementary to a nucleotide in a nucleotide position to be analyzed of the RNA sample is not ligated in step (c).

[0025] According to some embodiments, the RNA ligase is selected from the group consisting of: (i) KOD RNA ligase selected from KOD1Rnl from Thermococcus kodacryoides, which ligase (not commercially available to date) has shown unexpectedly reliable results, (ii) PBCV-1 DNA ligase / Chlorella virus DNA ligase, (iii) engineered ligases from the PBCV-1 family, and (iv) engineered ligases from the archaeal family Thermococcus kodacryoides.

[0026] The synthesis and characterization of this enzyme were disclosed in Zhang et al. (RNA Biol. 2017; 14(1): 36-44, “Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation”), where the template dependence and thermostability properties were discussed, as well as potential in vitro applications.

[0027] According to some embodiments, the detecting in step (e) is performed by luminescence, such as fluorescence, and / or by sequencing, such as sequencing by hybridization, sequencing by ligation, SOLiD sequencing or sequencing by synthesis.

[0028] Thus, analysis of single nucleotide variations in RNA samples can be easily obtained.

[0029] According to some embodiments, a detection oligonucleotide is added to the amplified circularized padlock oligonucleotide, wherein the detection oligonucleotide is designed to directly bind to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, or is added to a bridging oligonucleotide, which is designed to bind to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, so that the luminescent signal generated allows analysis of single nucleotide variations in the RNA sample.

[0030] According to some embodiments, by introducing different barcodes in the backbone sequence of the padlock probes for the competing PLP probes, the profiles of amplified circularized padlock oligonucleotides are distinguished by fluorescence readout.

[0031] As a result, the resolution and quality of the readout signal are further improved.

[0032] According to some embodiments, more than one single nucleotide variant located on the same RNA sample molecule is analyzed. Thus, multiple SNVs can be analyzed in the same experiment. In order to analyze multiple SNVs, a chimeric padlock probe designed for each SNV to be analyzed must be provided.

[0033] According to some embodiments, more than one single nucleotide variant located on different RNA sample molecules is analyzed. Thus, multiple SNVs / mutations in multiple transcript targets can be analyzed in a single experiment. A chimeric padlock probe designed for each SNV to be analyzed must be provided.

[0034] According to some embodiments, the terminal base at the 3' end of the chimeric padlock probe is selected from (i) an RNA base, (ii) a 2'-O-methoxy-ethyl base, (iii) a 2'-O-methyl RNA base, (iv) a 2'-fluoro base, (v) a DNA base, or (vi) an LNA base.

[0035] An "RNA base" is typically a normal RNA base, ie, unmodified A, G, C, or U, while a "DNA base" is typically a normal DNA base, ie, unmodified A, G, C, or T.

[0036] Therefore, using modifications at or near the 3' end of the padlock probe may improve ligase specificity and save costs, depending on the combination of the choice of modified base and the choice of ligase.

[0037] According to some embodiments, the modified base at the 3' end portion is positioned no more than two bases away from the 3' terminal base.

[0038] The "3' end portion" refers to the last approximately 2 to 3 bases located at the 3' end of the padlock probe.

[0039] According to some embodiments, the remaining bases of the 3' end portion are unmodified DNA bases.

[0040] According to some embodiments, the padlock probe has one of the following designs:

[0041] (i) the 3'-terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample;

[0042] (ii) the 3' terminal base is a modified base, and the base next to the 3' terminal base is located at the nucleotide position to be analyzed in the RNA sample; or

[0043] (iii) The base next to the 3' terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample.

[0044] According to some embodiments, the terminal base at the 5' end of the chimeric padlock probe is phosphorylated or pre-adenylated prior to hybridization with the RNA sample. Thus, the task of the ligase in the ligation step can be altered and, in some cases, facilitated and improved.

[0045] According to a second aspect, a kit for in situ analysis of single nucleotide variations in an RNA sample is provided, wherein the RNA sample comprises at least one nucleotide position to be analyzed, wherein the at least one nucleotide position to be analyzed is flanked on both the 3' side and the 5' side by nucleotide fragments with known identities, the kit comprising:

[0046] - one or more chimeric padlock oligonucleotides having a 3' end and a 5' end, wherein the 3' end and the 5' end are designed to hybridize to the nucleotide fragments with known identities at the 3' side and the 5' side of the RNA sample and the at least one nucleotide position to be analyzed, respectively, so that the 3' terminal base of the 3' end or any one of the two bases next to the 3' terminal base is designed to be located at the nucleotide position to be analyzed in the RNA sample when the padlock probe hybridizes to the RNA sample, and wherein the 3' terminal portion of the padlock oligonucleotide comprises a modified base at the 3' terminal portion, wherein the modified base is selected from the group consisting of C, A, G and U / T;

[0047] - an RNA ligase with single nucleotide specificity, optionally comprising necessary reagents and buffers;

[0048] -Optionally one or more amplification primers and polymerase for rolling circle amplification, as well as necessary reagents and buffers;

[0049] -Instruction manual.

[0050] Thus, provided is a kit comprising the necessary reagents and instructions for carrying out the methods described in this disclosure.

[0051] According to some embodiments, the kit comprises: (v) one or more fluorescent dyes and / or one or more RNA base or modified base specific detection oligonucleotides.

[0052] According to some embodiments, the chimeric padlock oligonucleotide comprises an anchor sequence, and the kit further comprises means for immobilizing the chimeric padlock oligonucleotide via the anchor sequence.

[0053] According to some embodiments, the KOD RNA ligase is a KOD ligase selected from KOD1 Rn1 from Thermococcus kodacryoides.

[0054] According to some embodiments, chimeric padlock oligonucleotides are designed to analyze more than one single nucleotide variation located on the same or different RNA sample molecules.

[0055] According to some embodiments, the modified base of the 3' end portion of the chimeric padlock probe is selected from (i) RNA base, (ii) 2'-O-methoxy-ethyl base, (iii) 2'-O-methyl RNA base, (iv) 2'-fluoro base, (v) DNA base or (vi) LNA base.

[0056] According to some embodiments, the modified base at the 3' end portion is positioned no more than two bases away from the 3' terminal base.

[0057] According to some embodiments, the padlock probe has one of the following designs:

[0058] (i) the 3'-terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample;

[0059] (ii) the 3' terminal base is a modified base, and the base next to the 3' terminal base is located at the nucleotide position to be analyzed in the RNA sample; or

[0060] (iii) The base next to the 3' terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample.

[0061] According to some embodiments, the 5' terminal base at the 5' end is phosphorylated or pre-adenylated.

[0062] Therefore, the methods described in this disclosure provide at least the following novel advantages and features:

[0063] ●Previously, there was no reliable solution for in situ SNV detection in RNA.

[0064] Compared with methods requiring cDNA synthesis, this method can achieve approximately 5 times higher detection efficiency.

[0065] ●This method is compatible with FFPE samples, while cDNA-ISS (in situ sequencing) is difficult to be compatible with FFPE samples.

[0066] • Through the present disclosure, applications as solutions for further immuno-oncology research and diagnosis can be provided.

[0067] The effects and features of the second aspect are largely similar to those described above in conjunction with the first aspect. The embodiments mentioned in relation to the first aspect are largely compatible with the second aspect.

[0068] The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the present disclosure only by way of illustration. Those skilled in the art will understand that changes and modifications can be made within the scope of the present disclosure based on the guidance in the detailed description.

[0069] Therefore, it should be understood that the disclosure disclosed herein is not limited to the specific components of the described devices or the steps of the described methods, as such devices and methods can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive. It should be noted that, unless the context clearly dictates otherwise, the articles "a", "an", "the" and "said" used in the specification and the appended claims are intended to indicate the presence of one or more elements. Thus, for example, reference to "a unit" or "the unit" may include several devices, etc. In addition, the words "comprise", "include", "contain" and similar expressions do not exclude other elements or steps.

[0070] definition

[0071] In the context of "padlock probes", the term "chimeric" refers to probes comprising portions of different origins, such as nucleic acid probes containing both DNA and RNA bases, for example DNA probes with the 3' end substituted with RNA bases or 2-o-methyl RNA bases.

[0072] The term "in situ analysis" means measuring the abundance of variants (such as SNVs) in a biological sample.

[0073] The term "sample" should be interpreted as any biological tissue sample from any species, and / or cultured cells, for example on a microscope slide or coverslip.

[0074] The term "ISS" is an abbreviation for "in situ sequencing."

[0075] The term "PLP" is an abbreviation for "Padlock Probe".

[0076] The term "RCA" is an abbreviation for "rolling circle amplification."

[0077] The terms "SNP" and "SNV" are abbreviations for "single nucleotide polymorphism" and "single nucleotide variation," respectively. Both terms refer to a variation / mutation at a single nucleotide position compared to a reference sequence (e.g., wild type). In the context of the present disclosure, the terms "SNP" and "SNV" are used interchangeably.

[0078] The term "LNA" refers to locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and commonly referred to as inaccessible RNA, which can be a modified RNA or DNA nucleotide in which the ribose moiety is modified with an additional bridge connecting the 2' oxygen and the 4' carbon. This bridge "locks" the ribose in the 3'-endo (North) conformation, which is typically found in A-form duplexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above objects and other objects, features and advantages of the present disclosure will be more fully understood by referring to the following illustrative and non-limiting detailed description of example embodiments of the present disclosure in conjunction with the accompanying drawings.

[0080] Figure 1 An example of a padlock probe using cDNA as a template according to prior art methods is disclosed.

[0081] Figure 2 A cDNA-based ISS (in situ sequencing) method for SNP analysis was demonstrated (Gyllborg et al., “Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue,” Nucleic Acids Research, Vol. 48, No. 19, November 4, 2020, p. e112, https: / / doi.org / 10.1093 / nar / gkaa792 ).

[0082] Figure 3 A comparison of a cDNA-based (1) and an RNA-based (2) approach according to the present disclosure is shown.

[0083] Figure 4 Shown is a comparison of a cDNA-based ISS (left) and an RNA-based ISS according to the present disclosure (right).

[0084] Figure 5 The principles of one embodiment of the present disclosure are shown, including competitive chimeric padlock probes and subsequent detection.

[0085] Figure 6 (A) discloses the overall design of the chimeric padlock probe used in the present disclosure. Its hybridization to an mRNA template is shown in (B).

[0086] Figures 7 to 10 Single nucleotide variation (SNV) detection using KOD ligase is shown.

[0087] Figures 11 to 14 Shown are competing padlock probes on known GPDH sequences in A549 cells.

[0088] Figure 15 An alternative design of a padlock probe comprising a 5' modification is shown.

[0089] Figure 16 shows an alternative design of a padlock probe comprising a 3' modification. DETAILED DESCRIPTION

[0090] The present inventors have developed a method comprising a method and a kit that uses a chimeric padlock probe in combination with a single nucleotide specific ligase to directly detect single nucleotide variations / polymorphisms on RNA molecules. This method thus solves the problem of improved spatial resolution and detection sensitivity compared to traditional cDNA-based methods. Reference will now be made to Figure 1 6 to describe the general principle of the present method compared with prior art solutions.

[0091] Figure 1 Examples of padlock probes in prior art methods are disclosed, wherein the padlock probes are designed to hybridize with cDNA molecules generated by reverse transcription from mRNA molecules of interest. Typical components of a padlock probe are shown, including a 5' arm for hybridizing with the template molecule at the 5' side of the position to be identified, an ID sequence and anchor sequence serving as a template for rolling circle amplification, and a 3' arm for hybridizing with the template molecule at the 3' side of the position to be identified. The 3' arm contains RNA bases at its end for ligation purposes. A bridging probe and a readout detection probe are designed to hybridize with the amplified probe for detection purposes.

[0092] Figure 2 cDNA-based ISS (in situ sequencing) is shown, including the necessary steps from (A) mRNA extraction, reverse transcription, PLP hybridization, ligation and rolling circle amplification to (B) and (C) detection in continuous cycles enabling analysis of multiple rolling circle products.

[0093] Figure 3 A comparison of cDNA-based ISS and the newly developed direct mRNA targeting method for SNP analysis in this disclosure is presented.

[0094] 1) Schematic diagram of cDNA-based ISS, in which cDNA is first synthesized in situ from mRNA before native mRNA digestion, allowing the use of a competitive DNA padlock probe (PLP) to probe the single-stranded cDNA. The correct PLP is then hybridized to the cDNA before a 100% matching PLP ligation event, followed by amplification by rolling circle amplification.

[0095] 2) Schematic representation of the improved chemistry whereby a competing chimeric PLP is directly hybridized to the mRNA in situ, followed by ligation of only the correct probes using KOD ligase, followed by RCA.

[0096] In a gesture Figure 1 and 2 In both, the resulting rolling circle amplification products can then be fluorescently labeled with fluorescent oligonucleotides, which can then be visualized under a microscope for fluorescent readout.

[0097] Figure 4 A comparison of a cDNA-based ISS (left) and an RNA-based ISS according to the present disclosure (right) is shown. Compared to the cDNA-based ISS, the RNA-based ISS omits one step (reverse transcription of mRNA into cDNA).

[0098] Figure 5 The principle of the method of the present disclosure is shown, wherein a competitive chimeric padlock probe is hybridized to an mRNA template, ligated (not shown), amplified (not shown), and subsequently detected by fluorescence. According to this embodiment, multiple single nucleotide variations in an RNA template molecule can be analyzed as long as the size of the padlock probe allows. Typically, the nucleotide distance between two single nucleotide variations on the same RNA molecule needs to be at least 30 nucleotides. As long as this criterion is met, there is no upper limit to the number of single nucleotide variations that can be analyzed in a single experiment.

[0099] Figure 6 (A) discloses the overall design of the chimeric padlock probe used in the present disclosure. (B) shows its hybridization to an mRNA template, and also shows that a single nucleotide variation (such as a point mutation) in the mRNA can be located approximately 0 to 6 nucleotides from the attachment point on the 3' end of the padlock probe.

[0100] Therefore, a first aspect of the present disclosure shows a method for in situ analysis of single nucleotide variations in an RNA sample, the method comprising the following steps:

[0101] (a) contacting an RNA sample with a plurality of chimeric padlock probes comprising an RNA base at the 3' end, wherein the RNA base is selected from the group consisting of C, A, G, and U, under conditions and reagents that permit hybridization;

[0102] (b) adding RNA ligase under conditions and reagents that allow ligation of the padlock probe with single nucleotide specificity to generate a circularized padlock oligonucleotide for the padlock probe comprising an RNA base complementary to a corresponding position of the RNA sample;

[0103] (c) amplifying the circularized padlock oligonucleotide under conditions and reagents that allow rolling circle amplification, thereby generating an amplified circularized padlock oligonucleotide;

[0104] (d) detecting the amplified circularized padlock oligonucleotides to obtain a profile of single nucleotides in the RNA sample.

[0105] RNA samples

[0106] In one embodiment, the RNA sample comprises at least one nucleotide position to be analyzed that is flanked on the 3' and 5' sides by nucleotide fragments with known identities.

[0107] In one embodiment, the RNA sample is an mRNA sample.

[0108] RNA samples can be obtained from any chosen tissue or cell line and, for example, can be pre-treated as exemplified in Example 1. Those skilled in the art will be aware of alternative methods of extracting and / or pre-treating sample material.

[0109] For example, tumor sections can be used to analyze mutations in the tumor microenvironment for immuno-oncology applications. However, a variety of other applications are also envisioned.

[0110] In order for an RNA sample to be used in the methods of the present disclosure, at least a portion of the RNA sample must contain a known nucleotide sequence so that a padlock probe can be designed that hybridizes to the RNA sample (as described below). At a minimum, a nucleotide fragment of the RNA sample corresponding to the length of the portion of the padlock probe designed to hybridize to the RNA sample (i.e., the 3' arm and the 5' arm) must be known. In addition, the RNA sample contains at least one nucleotide position to be analyzed, i.e., the position of the single nucleotide variation (or polymorphism) (SNV / SNP) of interest to be identified. Therefore, the SNV must be flanked on both sides by known sequences in order to design padlock probes that hybridize to the SNV at the 3' arm and the 5' end.

[0111] In one embodiment, the RNA sample contains at least two single nucleotide variations (SNVs) at different positions in the RNA sample. In this case, a different padlock probe must be designed for each SNV. The at least two SNVs can be (1) positioned at such a distance that the at least two different padlock probes designed for each SNV can hybridize simultaneously with the nucleotide regions flanking each SNV. In this case, the nucleotide distance between the two SNVs must be at least the length of the 3' arm of one of the padlock probes plus the length of the 5' arm of the other padlock probe, i.e., about 15+15 nucleotides. Otherwise, the at least two padlock probes cannot hybridize with the same RNA sample molecule at the same time. Alternatively, (2), the at least two SNVs can be positioned at a closer distance, which means that for each individual RNA molecule, only one of the at least two different padlock probes can hybridize, which means that the at least two different padlock probes will compete for hybridization with the RNA sample, which typically results in a lower detection signal. Therefore, if the SNVs are positioned too close (less than about 30 nucleotides apart), it is typically observed that the detection efficiency is lower when detecting two SNVs simultaneously.

[0112] Padlock Technology / Padlock Probes

[0113] In one embodiment, a plurality of chimeric padlock probes each have a first end and a second end designed to hybridize to nucleotide fragments with known identities at the 3' and 5' sides of an RNA sample and at least one nucleotide position to be analyzed, respectively, such that the RNA base at the 3' end is positioned at the nucleotide position to be analyzed in the RNA sample.

[0114] In one embodiment, a padlock probe having an RNA base at its 3' end that is complementary to the nucleotide in the nucleotide position to be analyzed of the RNA sample is ligated in step (c), and wherein a padlock probe having at least one RNA base at its 3' end that is not complementary to the nucleotide in the nucleotide position to be analyzed of the RNA sample is not ligated in step (c).

[0115] The overall design of the mating padlock probe is as follows Figure 1 and / or as shown in Figure 6. Techniques for designing chimeric padlock probes are generally known in the art.

[0116] In this context, the selection and design of padlock probes needs to take into account the SNV or SNVs to be analyzed. A number of different situations may arise that may alter the choice of padlock probe to be designed and used:

[0117] In one case, an RNA sample contains a SNV to be analyzed, wherein the identity of the SNV is unknown and can vary between all four bases (A, G, C, U). In this case, four padlock probes with the same overall design but different RNA bases at the 3' end are typically designed and used. When used, the padlock probe with an RNA base complementary to the SNV of the RNA sample to be identified will fully hybridize with the RNA sample (including the 3' end) (i.e., U hybridizes with A, A hybridizes with U, C hybridizes with G, and G hybridizes with C), and is subsequently connected, amplified, and detected. Other padlock probes that contain RNA bases that are not complementary to the SNV of the RNA sample at the 3' end will typically hybridize (at least partially hybridize), but will include mismatches at the SNV position and will therefore not be connected, amplified, and detected. Therefore, detection will only occur for padlock probes that contain RNA bases that are base-paired with the SNV at the 3' end, and the identity of the SNV will therefore be determined.

[0118] In another case, the RNA sample contains a SNV to be analyzed, wherein the identity of the SNV is partially unknown and is expected to vary between less than four bases (e.g., two bases). In this case, two padlock probes with the same overall design but different RNA bases at the 3' end are typically designed and used. The padlock probe designed and used will include RNA bases complementary to the expected identity of the SNV at the 3' end, i.e., for example, if the SNV is expected to be A or G, the designed padlock probe will include RNA bases U and C at the 3' end. In this case, there is no need to design a padlock probe containing RNA bases that are not expected to be complementary to the SNV, and costs can be saved. When used, the padlock probe with RNA bases complementary to the SNV of the RNA sample to be identified will fully hybridize with the RNA sample (including the 3' end) (i.e., U hybridizes with A, A hybridizes with U, C hybridizes with G, and G hybridizes with C), and is subsequently connected, amplified, and detected. Another padlock probe comprising an RNA base at its 3' end that is not complementary to the SNV of the RNA sample will typically hybridize (at least partially) but will include a mismatch at the SNV position and therefore will not be ligated, amplified, and detected.

[0119] In another case, the RNA sample may contain more than one SNV to be analyzed, and in this case, more than one padlock probe can be designed and used. The general principles of cases (1) and (2) can also be applied here. However, in order for different padlock probes to hybridize simultaneously with different SNVs on the same RNA sample molecule, the padlock probes must be designed so that the nucleotide lengths of the hybridization parts of the padlock probes (i.e., the 3' arm and the 5' arm) allow simultaneous binding, i.e., the 3' arm length of one padlock probe + the 5' arm length of another padlock probe is not shorter than the nucleotide distance between at least two SNVs.

[0120] In another scenario, where multiple SNVs / mutations are present across multiple transcript targets, a pool of wild-type and mutant chimeric padlock probes can be designed and used to simultaneously probe multiple SNVs. The signatures of these spatially analyzed mutations can then be extracted from a single experiment.

[0121] Details of typical conditions that allow padlock probes to hybridize to RNA samples are provided in the Examples section below.

[0122] In order to subsequently detect the identity of the SNV to be analyzed, a detection oligonucleotide can be used, which can be combined with a bridging oligonucleotide (or bridging probe). Typically, the padlock oligonucleotide is designed to contain an ID sequence that is specific for different padlock probes, depending on the identity of the RNA base at the 3' end, that is, for each possible alternative identity of the SNV site to be analyzed, the specific ID sequence is unique. Therefore, when used, a bridging oligonucleotide that is complementary to the ID sequence of the amplified circularized padlock oligonucleotide present will hybridize with the ID sequence. The detection oligonucleotide containing a fluorescent signal molecule can then be detected, which provides a specific signal for the RNA base at the 3' end of the connected padlock probe (thereby identifying SNVs in the RNA sample).

[0123] In some embodiments, the padlock oligonucleotides also contain an anchor sequence for entry into the amplification event, thereby enabling rapid quality control.

[0124] The Examples section shows an example of typical conditions for padlock hybridization according to the method of the present invention, wherein PLP hybridization is performed overnight at 37°C (the temperature can be varied between about 37 and 55°C). Hybridization may or may not be followed by a wash step to eliminate excess unhybridized probe. In addition, an additional blocking reagent, such as salmon sperm DNA, can be added as an optional step. The salmon sperm DNA solution was developed for use as a blocking reagent in hybridization protocols to reduce nonspecific binding of hybridization probes ( https: / / assets.fishersci.com / TFS-Assets / LSG / manuals / 15632011.pdf For the same purpose, an alternative blocking reagent for salmon sperm DNA is yeast tRNA ( https: / / assets.thermofisher.com / TFS-Assets%2FLSG%2Fmanuals%2Fsp_7119.pdf ).

[0125] Modifications and alternative designs of padlock probes

[0126] In some padlock probe designs, the 3' and / or 5' ends of the padlock probe can be modified. Modifying the ends of the padlock probe can generally improve single nucleotide specificity. Furthermore, by using ligase-accepted base modifications for SNP analysis, probe costs can be reduced. Furthermore, ligation efficiency can be improved.

[0127] Regarding 5' modification ( Figure 15), the 5' end of the probe used can be pre-adenylated prior to hybridization with the RNA sample, rather than having a 5' phosphate group. Typically, PLP is provided 5'-phosphorylated. However, ligation is performed in two steps: first, the ligase adenylates the 5' end, and then catalyzes the formation of a phosphodiester bond. The inventors have improved this method by first adenylylating the probe using a 5' adenylylation kit, and then hybridizing these adenylylated probes. This allows the ligase to catalyze only one step, rather than two, potentially improving the efficiency of the ligation and, therefore, the overall method.

[0128] For 3' modification (Figure 16(ac)), the following alternatives are provided:

[0129] (a) When the mutant / wild-type (nucleotide to be analyzed) base is at the 3' end (position n) of the probe, the 3' end base (position n) modification can be:

[0130] RNA base (A, G, C, or U)

[0131] 2'-O-methoxyethyl base (2'-MOE)

[0132] 2'-O-methyl RNA bases

[0133] 2' fluoro base

[0134] Unmodified DNA bases

[0135] LNA bases

[0136] (b) When the mutant / wild-type (nucleotide to be analyzed) base is located at position (n-1) next to the 3' end of the probe, the terminal base at 3' (position n) has the following base modification:

[0137] RNA base (A, G, C, or U)

[0138] 2'-O-methoxyethyl base (2'-MOE)

[0139] 2'-O-methyl RNA bases

[0140] 2' fluoro base

[0141] Unmodified DNA bases

[0142] LNA bases

[0143] (c) When the mutant / wild-type (nucleotide to be analyzed) base is located at the position (position n) next to the 3' end (n-1) of the probe, the base at position (n-1) has the following base modification:

[0144] RNA base (A, G, C, or U)

[0145] 2'-O-methoxyethyl base (2'-MOE)

[0146] 2'-O-methyl RNA bases

[0147] 2' fluoro base

[0148] Unmodified DNA bases

[0149] LNA bases

[0150] (d) The mutant / wild-type (nucleotide to be analyzed) base is located two bases away from the 3' end of the probe (n-2)

[0151] The mutant / wild-type base at position (n-2) has the following base modifications:

[0152] RNA base (A, G, C, or U)

[0153] 2'-O-methoxyethyl base (2'-MOE)

[0154] 2'-O-methyl RNA bases

[0155] 2' fluoro base

[0156] Unmodified DNA bases

[0157] LNA bases

[0158] In embodiments where a base is at position (n-1) or (n-2), the base at position n or at positions n and (n-1), respectively, is typically a standard DNA base. Typically, positions of the probe that do not have unmodified bases are typically standard / normal DNA bases.

[0159] The probe modifications listed above have been shown to be compatible with the function of the ligases used and suggested in this disclosure for SNP detection purposes. Some of these probe modifications represent probe alternatives not previously disclosed for such applications.

[0160] LNA bases have the potential advantage that LNA can provide improved base pairing specificity, which will allow correct probes to hybridize better than mismatched probes, thereby helping the ligase to distinguish between perfect hybridization and imperfect hybridization before ligation.

[0161] connect

[0162] A key feature of the present invention is the use of RNA ligase with single nucleotide specificity. Thus, the specificity of RNA ligase enables the distinction between padlock probes with mismatches at the SNV (i.e., the RNA base at its 3' end is not complementary to the SNV nucleotide identity) and padlock probes that are base-paired with the SNV position (i.e., the RNA base at the 3' end is complementary to the SNV nucleotide identity). For padlock probes that are base-paired with the SNV position, RNA ligase will block the 3' and 5' ends of the padlock oligonucleotide, thereby generating a circularized padlock oligonucleotide, which is then amplified and detected to determine the identity of the SNV position.

[0163] Single nucleotide specificity can be verified, for example, by a validation model based on a sequencing database, targeting known isomers / SNPs / SNVs (single nucleotide polymorphisms / variants). In addition, the specificity of the methods disclosed in this specification can be verified by running control experiments using cDNA-ISS, which has previously been shown to be able to analyze mutations on cDNA generated in situ from mRNA. The results obtained correlate well with cDNA-ISS data.

[0164] In some embodiments, single nucleotide specificity can be quantified where only 100% correctly matched hybridizations of the padlock probes need to be ligated. Various modifications at the 3' end, such as those listed in this disclosure, can facilitate the ligase in distinguishing between perfectly (100%) hybridized constructs and mismatched (less than 100%) hybridizations before ligation occurs.

[0165] In one embodiment, the RNA ligase is a KOD RNA ligase selected from KOD1 Rn1 from Thermococcus kodacrya. This RNA ligase has been shown to be able to ligate padlock probes with sufficiently high accuracy.

[0166] An important criterion for selecting a ligase that may function in the methods of the present disclosure is the ability of the ligase to accept RNA as a scaffold in order to catalyze the ligation reaction.

[0167] In some embodiments, the ligase is selected from PBCV-1 DNA ligase / Chlorella virus DNA ligase, an engineered ligase from the PBCV-1 family, and an engineered ligase from the archaeal family Thermococcus kodacrya.

[0168] For the typical conditions that allow to connect with KOD RNA ligase, details are provided in the example section below.Certainly, condition can change according to the selection of ligase.Usually, the selection of ligase will determine other necessary conditions.Before the selection of ligase, monovalence and divalent salt concentration and pH are the most important parameters.For KOD ligase, it is reported that optimum pH is about 7.5, and therefore the pH in about 7 to 8 intervals is suitable for ligation step usually.

[0169] Rolling circle amplification

[0170] The circularized padlock probe, ie, the padlock probe that has undergone ligation and thus contains an RNA base at the 3' end position of the ligation that is complementary to the position of the SNV to be identified, will undergo amplification, typically using rolling circle amplification.

[0171] Details regarding typical conditions that allow for rolling circle amplification are provided in the Examples section below.

[0172] In some embodiments, primers, enzymes, and other reagents for the hybridization, ligation, and amplification steps (typically RCA) can be added in stages, or at least partially combined, to improve the efficiency of the method. For example, an RCA primer can be added to the ligation mixture and allowed to anneal to the hybridized chimeric PLP. Additionally, a competing PLP can be added to the ligation mixture.

[0173] Detection

[0174] Detection of the amplified circularized padlock probes can be performed in several different ways to analyze one or more single nucleotide variations (SNVs) to be identified, for example by luminescence (such as fluorescence) and / or by sequencing (such as sequencing by hybridization (SBH), sequencing by ligation (SBL), SOLiD sequencing or sequencing by synthesis (SBS)).

[0175] In one embodiment, a detection oligonucleotide comprising means for luminescent signal transmission is added to the amplified circularized padlock oligonucleotide, wherein the detection oligonucleotide is designed to bind directly to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, or is added to a bridging oligonucleotide, which is designed to bind to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, so that the luminescent signal generated allows analysis of single nucleotide variations in RNA samples.

[0176] In one embodiment, the bridging oligonucleotide is specific for the ID sequence contained in the padlock probe. The ID sequence is specific for different padlock probes, specifically depending on the identity of the RNA base at the 3 ends (i.e., whether the RNA base is U, A, C or G), and therefore only the bridging oligonucleotide corresponding to the specific RNA base at the 3 ends of the padlock oligonucleotide to which it is hybridized will hybridize. In this way, only the amplified cyclized oligonucleotide is likely to be detected. Generally, this means that in order to obtain a detectable signal, bridging oligonucleotides corresponding to all possible SNV variations should be added. For example, in the case of four (4) different SNV variations (A, C, U, G), this means that four (4) different bridging oligonucleotides should be added.

[0177] In addition, a detection oligonucleotide specific for each alternative bridging oligonucleotide is added, wherein each detection oligonucleotide contains a luminescent (such as fluorescent) signal transmission molecule. After the specific detection oligonucleotide is bound to the bridging oligonucleotide to which it is specific, the bridging oligonucleotide will hybridize specifically with the amplified circularized padlock oligonucleotide according to the identity of the RNA base at the 3' end (and therefore according to the identity of the SNV to be analyzed), and a unique detection signal for the identity of the SNV to be analyzed can be detected. Generally, this means that in order to obtain a detectable signal, detection oligonucleotides corresponding to all available bridging oligonucleotides should be added. For example, in the case of four (4) different bridging oligonucleotides available, four (4) different detection oligonucleotides that provide different detection signals should be added.

[0178] In cases where the RNA sample contains more than one SNV to be analyzed, detection can be performed by adding an additional, unique set of detection oligonucleotides and / or bridging oligonucleotides to provide additional detection signals that enable differentiation of different SNVs in addition to analyzing the identity of each SNV. Alternatively, detection can be performed in cycles, where only the identity of one SNV is analyzed for each cycle.

[0179] Details on various options and alternatives in this regard can be found in the following references: https: / / doi.org / 10.1093 / nar / gkaa792 .

[0180] Thus, luminescent signaling may include a fluorescent signal that is detectable via, for example, microscopy or other imaging techniques allowing detection of the fluorescent signal.

[0181] In one embodiment, by introducing different barcodes in the backbone sequence of the padlock probe for the competing PLP probe, the patterns of amplified circularized padlock oligonucleotides can be distinguished by fluorescence readout. The backbone sequence design of the competing PLP (where the RNA base at the 3' end can be A, U, C, or G) can have a unique backbone sequence that can be distinguished by the fluorescent detection probe after RCA.

[0182] Reagent test kit

[0183] In a second aspect of the present disclosure, there is provided a kit for in situ analysis of single nucleotide variations in an RNA sample according to the first aspect, wherein the sample comprises at least one nucleotide position to be analyzed, and the at least one nucleotide position to be analyzed is flanked by nucleotide fragments with known identities on both the 3' side and the 5' side, the kit comprising:

[0184] - one or more chimeric padlock oligonucleotides having a 3' end and a 5' end, wherein the 3' end and the 5' end are designed to hybridize to the nucleotide fragments of known identity at the 3' side and the 5' side of the RNA sample and the at least one nucleotide position to be analyzed, respectively, such that the 3' terminal base of the 3' end or the base next to the 3' terminal base is designed to be positioned at the nucleotide position to be analyzed in the RNA sample when the padlock probe hybridizes to the RNA sample, and wherein the 3' terminal portion of the padlock oligonucleotide comprises a modified base at the 3' terminal portion, wherein the modified base is selected from the group consisting of C, A, G and U / T;

[0185] - an RNA ligase with single nucleotide specificity, optionally comprising necessary reagents and buffers;

[0186] -Optionally one or more amplification primers and polymerase for rolling circle amplification, as well as necessary reagents and buffers;

[0187] -Instruction manual.

[0188] In one embodiment, the kit comprises: (v) one or more fluorescent dyes and / or one or more RNA base-specific detection oligonucleotides.

[0189] In one embodiment, the chimeric padlock oligonucleotide comprises an anchor sequence, and the kit further comprises means for immobilizing the chimeric padlock oligonucleotide via the anchor sequence.

[0190] In one embodiment, the RNA ligase is selected from the group consisting of: (i) a KOD ligase selected from KOD1 Rn1 from Thermococcus kodachii, (ii) PBCV-1 DNA ligase / Chlorella virus DNA ligase, (iii) an engineered ligase from the PBCV-1 family, and (iv) an engineered ligase from the archaeal family Thermococcus kodachii.

[0191] In one embodiment, chimeric padlock oligonucleotides are designed to analyze more than one single nucleotide variation located on the same or different RNA sample molecules.

[0192] In one embodiment, the modified base of the 3' end portion of the chimeric padlock probe is selected from (i) RNA bases (selected from A, G, C and U), (ii) 2'-O-methoxy-ethyl bases, (iii) 2'-O-methyl RNA bases, (iv) 2'-fluoro bases, (v) DNA bases or (vi) LNA bases.

[0193] In one embodiment, the modified base at the 3' end portion is positioned no more than two bases away from the 3' terminal base.

[0194] In one embodiment, the padlock probe has one of the following designs:

[0195] (i) the 3'-terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample;

[0196] (j) the 3'-terminal base is a modified base, and the base next to the 3'-terminal base is located at the nucleotide position to be analyzed in the RNA sample; or

[0197] (k) The base next to the 3' terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample.

[0198] In one embodiment, the 5' terminal base at the 5' end is phosphorylated or pre-adenylated.

[0199] Thus, kits for use with the methods of the present disclosure generally contain the reagents required for the overall method, and often contain specific probes and materials for particular analytical applications.

[0200] The present invention will be described by the following examples, which are intended to illustrate specific embodiments of the present invention but should not be construed as limiting the present invention in any way.

[0201] Examples

[0202] The present disclosure will be described with reference to the accompanying examples, which illustrate preferred exemplary embodiments of the disclosure. However, the present disclosure may be embodied in other forms and should not be construed as being limited to the embodiments disclosed herein. The disclosed embodiments are provided to fully convey the scope of the present disclosure to those skilled in the art.

[0203] Example 1: SNV detection protocol using KOD ligase and chimeric probes

[0204] It will be appreciated by those skilled in the art that certain reagents and conditions may be modified. Therefore, the examples provided are intended only to illustrate the present invention.

[0205] Sample pretreatment

[0206] Fresh frozen biological samples (can be cell lines / any tissue that has been sectioned onto microscope slides / cover slips) are first fixed with 3.7% formaldehyde.

[0207] If the sample is an FFPE sample, it is first deparaffinized / decrosslinked with xylene and heat treated (ie, incubated at 45°C for 15 minutes).

[0208] The biological sample is then permeabilized with 0.1 M HCl and pepsin or proteinase K, or any other permeabilization reagent commonly used in FISH experiments, is added.

[0209] Chimeric lock probe design (see Figure 5 A, Understanding universal chimeric PLP design).

[0210] First, a chimeric PLP is designed to contain two arms (arm 1 and arm 2) that are complementary to the mRNA sequence of interest. The combined length of arm 1 and arm 2 should be 30 to 50 nt (for a symmetrical design, each arm should be 15 to 25 nt long). Asymmetric PLPs can also be designed with a longer 5' arm (arm 1) and a shorter 3' arm (arm 2).

[0211] The presence of an RNA base (rA, rU, rC, or rG) at the 2-terminal base of the 3' arm is an essential feature for the correct functioning of certain RNA ligases.

[0212] The user can introduce two (2) or more unique backbone sequences, including an RCA priming site to allow RCA primers to anneal, and any other unique sequences to aid downstream probe recognition after amplification.

[0213] Design of chimeric PLPs using KOD ligase analysis of mutations ( Figure 5 B):

[0214] To analyze point mutations / SNPs / SNVs in situ on the mRNA of interest, PLPs can be designed such that the point mutation is located at the 3' end of the PLP within a length range of 0 to 6 nt from the junction nick.

[0215] hybridization

[0216] Chimeric padlock probes (PLPs) are then added to the hybridization buffer to perform PLP hybridization.

[0217]

[0218] Table 1. Hybridization mixture.

[0219] PLP hybridization is performed overnight at 37°C (temperature can be adjusted between 37 and 55°C). Hybridization may or may not be followed by a wash step to eliminate excess unhybridized probe. Optionally, an additional blocking reagent, such as salmon sperm DNA or yeast tRNA, may be added.

[0220] connect

[0221] 1X KOD ligation buffer 2mM DTT 50mM NaCl 50mM Tris-HCl <![CDATA[10mM MgCl2]]> 0.8 mM ATP 0.05 μM RCA primer 1U / μl RiboProtect (RNase inhibitor) 0.1 μg / μl KOD ligase

[0222] Table 2. Examples of KOD ligase ligation buffer compositions and conditions.

[0223] RCA primers (which can also be added to the RCA mix below) are also added to the ligation mix to anneal to the hybridized chimeric PLP. Competing PLP can also be added to the ligation mix. Ligation is performed at 55°C for 2 hours.

[0224] The pH during the ligation step should be in the range of 7 to 8.

[0225] Rolling circle amplification (RCA)

[0226]

[0227]

[0228] Table 3. RCA reaction buffer composition.

[0229] RCA primers can also be added to the RCA mix if they have not been added to the ligation mix previously.

[0230] Example 2 - Competitive Padlock Probe Detection of Known SNVs on GAPDH

[0231] Figures 7 to 10 Detection of single nucleotide variations using KOD ligase is shown.

[0232] Figure 7 Four competitive probes were included in the assay, each containing "A," "U," "C," and "G" at the 3' end. The SNV to be analyzed was "U," meaning the only padlock probe that would be ligated and produce a detectable signal would be the one containing "A" at the 3' end. A detectable signal was obtained in the detection channel corresponding to the "A" probe (i.e., AF488), but not in any other channels.

[0233] exist Figure 8 A negative control was performed. Only padlock probes without an "A" at the 3' end were included. The SNV to be analyzed was "U," meaning no padlock probes were expected to ligate and generate a detectable signal. As can be seen, no detectable signal was obtained in either the AF488 channel or any other channel.

[0234] Figure 9 Four competitive probes were included in the assay, each containing an "A," "U," "C," and "G" at the 3' end. The SNV to be analyzed was "C," meaning the only padlock probe that would be ligated and produce a detectable signal would be the one containing a "G" at the 3' end. A detectable signal was obtained in the detection channel corresponding to the "G" probe (i.e., Atto425), but not in any other channels.

[0235] exist Figure 10 A negative control was performed. Only padlock probes without a "G" at the 3' end were included. The SNV to be analyzed was "C," meaning no padlock probes were expected to be ligated. As can be seen, no detectable signal was obtained in either the Atto425 channel or any other channel.

[0236] Example 3 - Competitive Padlock Probe for Known GAPDH Sequence in A549 Cells

[0237] Figures 11 to 14 Shown is the performance of competing padlock probes on known GAPDH sequences in A549 cells.

[0238] Figure 11 Four competitor probes are included in the assay, each containing "A," "U," "C," and "G" at the 3' end. The SNV to be analyzed is "C," which means that the only padlock probe that will be ligated and produce a detectable signal should be the probe containing "G" at the 3' end. Figure 12 Detection results are disclosed, wherein only the channel of the "G" padlock probe provides a detectable signal.

[0239] Figure 13 Two competitive padlock probes, "A" and "G," are included in the assay. The RNA sample (GAPDH) contains two alleles of the SNP to be detected, one allele being "T" and the other allele being "C," located at the position to be analyzed. Figure 14 The published test results show that the "T" allele appears at a higher frequency, and that the "C" allele appears but at a lower frequency.

[0240] Those skilled in the art will appreciate that the present disclosure is not limited to the preferred embodiments described above. Those skilled in the art will also appreciate that modifications and variations are possible within the scope of the appended claims. Furthermore, those skilled in the art will understand and implement variations of the disclosed embodiments when practicing the claimed disclosure by studying the drawings, the disclosure, and the appended claims.

Claims

1. A method for in situ analysis of single nucleotide variations in an RNA sample, comprising the following steps: (a) contacting the RNA sample with a plurality of chimeric padlock probes under conditions and reagents that allow hybridization, wherein the chimeric padlock probes comprise a modified base at the 3' end portion, wherein the modified base is selected from the group consisting of C, A, G, and U / T; (b) adding RNA ligase under conditions and reagents that allow ligation of the padlock probe with single nucleotide specificity to generate a circularized padlock oligonucleotide for the padlock probe comprising a modified base complementary to a corresponding position of the RNA sample; (c) amplifying the circularized padlock oligonucleotide under conditions and reagents that allow rolling circle amplification, thereby generating an amplified circularized padlock oligonucleotide; (d) detecting the amplified circularized padlock oligonucleotides to obtain a profile of single nucleotides in the RNA sample.

2. The method according to claim 1, wherein the RNA sample comprises at least one nucleotide position to be analyzed, and the at least one nucleotide position to be analyzed is flanked on the 3' side and the 5' side by nucleotide fragments with known identities.

3. The method according to claim 1 or 2, wherein the RNA sample is selected from an mRNA sample, an rRNA sample, a microRNA sample or a non-coding RNA sample.

4. The method according to claim 2 or 3, wherein each of the plurality of chimeric padlock probes has a first end and a second end, and the first end and the second end are designed to hybridize with the nucleotide fragments with known identities at the 3' side and the 5' side of the RNA sample and the at least one nucleotide position to be analyzed, respectively, so that the terminal base at the 3' end of the padlock probe or any one of the two bases next to the terminal base at the 3' end is positioned at the nucleotide position to be analyzed in the RNA sample.

5. The method according to any one of claims 2 to 4, wherein the padlock probe having a base at the 3' end portion that is complementary to the nucleotide in the nucleotide position to be analyzed of the RNA sample is ligated in step (c), and wherein the padlock probe having at least one base at the 3' end portion that is not complementary to the nucleotide in the nucleotide position to be analyzed of the RNA sample is not ligated in step (c).

6. The method according to any one of the preceding claims, wherein the RNA ligase is selected from the group comprising: (i) a KOD ligase selected from KOD1 Rn1 from Thermococcus kodachii, (ii) PBCV-1 DNA ligase / Chlorella virus DNA ligase, (iii) an engineered ligase from the PBCV-1 family, and (iv) an engineered ligase from the archaeal family Thermococcus kodachii.

7. The method according to any one of the preceding claims, wherein the detection in step (e) is performed by luminescence, such as fluorescence, and / or by sequencing, such as sequencing by hybridization (SBH), sequencing by ligation (SBL), SOLiD sequencing or sequencing by synthesis (SBS).

8. A method according to claim 7, wherein a detection oligonucleotide is added to the amplified circularized padlock oligonucleotide, and the detection oligonucleotide is designed to directly bind to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, or is added to a bridging oligonucleotide, and the bridging oligonucleotide is designed to bind to the amplified circularized padlock oligonucleotide in an RNA base-specific manner, so that the luminescent signal generated allows analysis of the single nucleotide variation in the RNA sample.

9. The method according to any one of the preceding claims, wherein the profiles of the amplified circularized padlock oligonucleotides are distinguished by fluorescence readout by introducing a different barcode to the competing PLP probe in the backbone sequence of the padlock probe.

10. The method according to any one of the preceding claims, wherein more than one single nucleotide variation located on the same RNA sample molecule is analyzed.

11. The method according to any one of the preceding claims, wherein more than one single nucleotide variation located on different RNA sample molecules is analyzed.

12. The method according to any one of the preceding claims, wherein the modified base at the 3' end portion of the chimeric padlock probe is selected from: (i) RNA base, (ii) 2'-O-methoxy-ethyl base, (iii) 2'-O-methyl RNA base, (iv) 2'-fluoro base, (v) DNA base or (vi) LNA base.

13. The method according to any one of the preceding claims, wherein the modified base at the 3' end portion is positioned no more than two bases away from the 3' terminal base. The method according to claim 13 , wherein the remaining bases of the 3′ end portion are unmodified DNA bases.

15. The method according to any one of the preceding claims, wherein the padlock probe has any one of the following designs: (i) the 3'-terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample; (ii) the 3'-terminal base is a modified base, and the base next to the 3'-terminal base is located at the nucleotide position to be analyzed in the RNA sample; or (iii) The base next to the 3' terminal base is a modified base, and is located at the nucleotide position to be analyzed in the RNA sample.

16. The method according to any one of the preceding claims, wherein the terminal base at the 5' end of the chimeric padlock probe is phosphorylated or pre-adenylated prior to hybridization with the RNA sample.

17. A kit for in situ analysis of single nucleotide variations in an RNA sample according to any one of claims 1 to 16, wherein the RNA sample comprises at least one nucleotide position to be analyzed, and the at least one nucleotide position to be analyzed is flanked on both the 3' side and the 5' side by nucleotide fragments with known identities, the kit comprising: a. one or more chimeric padlock oligonucleotides having a 3' end and a 5' end, wherein the 3' end and the 5' end are designed to hybridize to the nucleotide fragments with known identities at the 3' side and the 5' side of the RNA sample and the at least one nucleotide position to be analyzed, respectively, so that the 3' terminal base of the 3' end or any one of the two bases next to the 3' terminal base is designed to be positioned at the nucleotide position to be analyzed in the RNA sample when the padlock probe hybridizes to the RNA sample, and wherein the 3' end portion of the padlock oligonucleotide comprises a modified base at the 3' end portion, wherein the modified base is selected from the group consisting of C, A, G and U / T; b. an RNA ligase having single nucleotide specificity, optionally comprising necessary reagents and buffers; c. Optional one or more amplification primers and polymerase for rolling circle amplification, as well as necessary reagents and buffers; d.Instructions for use.

18. The kit of claim 17, further comprising: (v) one or more fluorescent dyes and / or one or more RNA base-specific detection and / or bridging oligonucleotides.

19. The kit according to any one of claims 17 or 18, wherein the chimeric padlock oligonucleotide comprises an anchor sequence, and the kit further comprises means for immobilizing the chimeric padlock oligonucleotide via the anchor sequence.

20. The kit according to any one of claims 17 to 19, wherein the RNA ligase is selected from the group comprising: (i) a KOD ligase selected from KOD1 Rn1 from Thermococcus kodacrya, (ii) PBCV-1 DNA ligase / Chlorella virus DNA ligase, (iii) an engineered ligase from the PBCV-1 family, and (iv) an engineered ligase from the archaeal family Thermococcus kodacrya.

21. The kit according to any one of claims 17 to 20, wherein the chimeric padlock oligonucleotides are designed to analyze more than one single nucleotide variation located on the same or different RNA sample molecules.

22. The kit according to any one of claims 17 to 21, wherein the modified base of the 3' end portion of the chimeric padlock probe is selected from: (i) RNA base, (ii) 2'-O-methoxy-ethyl base, (iii) 2'-O-methyl RNA base, (iv) 2'-fluoro base, (v) DNA base or (vi) LNA base. 23 . The kit according to claim 17 , wherein the modified base at the 3′ end portion is positioned no more than two bases away from the 3′ terminal base.

24. The kit according to any one of claims 17 to 23, wherein the padlock probe has any one of the following designs: (i) the 3'-terminal base is a modified base and is located at the nucleotide position to be analyzed in the RNA sample; (ii) the 3'-terminal base is a modified base, and the base next to the 3'-terminal base is located at the nucleotide position to be analyzed in the RNA sample; or (iii) The base next to the 3' terminal base is a modified base, and is located at the nucleotide position to be analyzed in the RNA sample.

25. The kit according to any one of claims 17 to 24, wherein the 5' terminal base of the 5' end is phosphorylated or pre-adenylated.

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