RNA-templated ligation

Abstract

The present application provides a method for detecting a target nucleic acid molecule in a sample, the method comprising: contacting the sample with a ligatable probe comprising one or more parts and hybridizing the probe with the target nucleic acid molecule, ligating any probe that has hybridized with the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby detecting the target nucleic acid molecule, wherein the probe comprises at least one ribonucleotide at or near the ligation site and / or wherein the probe or the probe portion comprises an additional sequence 5' of a target-specific binding site, the additional sequence 5' does not hybridize with the target nucleic acid molecule after the probe hybridizes with the target nucleic acid molecule and forms a 5' flap containing one or more nucleotides at its 3' end that is cut before ligation, and a method for synthesizing a DNA molecule using a template nucleic acid molecule comprising at least one ribonucleotide with a Phi29 DNA polymerase. A probe for use in a detection method is provided.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection. Specifically, the present invention provides a method for detecting a target nucleic acid sequence in a target nucleic acid, and in particular a target RNA sequence in a target RNA molecule, using a connectable chimeric DNA-RNA probe. Also provided is a nucleic acid probe for use in such a method. Background Art

[0002] Detection of target nucleic acid sequences has applications in many different areas, including personalized medicine, cancer diagnosis and treatment, infectious diseases, and biosafety.

[0003] Labeled hybridization probes complementary to target nucleic acid sequences can be used to detect target nucleic acid sequences, for example in Southern or Northern blot assays. Such probes allow simple and direct detection of target nucleic acid sequences (including target RNA sequences), but hybridization probes have a relatively high lower limit of detection and cannot be easily used to distinguish similar nucleic acid sequences. In addition, such probes cannot be easily adapted for multiple or automated detection assays.

[0004] The sensitivity of detecting target nucleic acid molecules can be enhanced by first amplifying the target sequence. Amplification techniques allow the amplification of the target sequence at a very low level in the sample before detection to increase the copy number of the target sequence in the sample. Amplification can be carried out using any of the various techniques available in the art, such as polymerase chain reaction (PCR), nucleic acid-specific amplification (NASBA), ligase chain reaction (LCR) and rolling circle amplification (RCA). However, these techniques are mainly directed to amplified DNA sequences, and typically require DNA templates to be effectively carried out.

[0005] PCR can be used to amplify a target nucleic acid molecule and allows exponential amplification using a pair of primers that are specific for sequences flanking the target nucleic acid sequence within the target nucleic acid molecule. However, the target RNA sequence cannot be detected directly by PCR, and instead detection of RNA requires the use of a reverse transcriptase (RT-PCR) to form a cDNA intermediate that can then be amplified. Similarly, NASBA and LCR also require an initial reverse transcriptase step in order to generate a cDNA template for amplification (CA 2136764).

[0006] RCA utilizes a strand displacement polymerase and requires a circular amplification template. Amplification of the circular template provides a cascade of RCA products comprising multiple copies of a sequence complementary to that of the amplification template, which copies can ultimately be detected. The circular RCA template is typically provided by a ligation reaction, and can typically be provided by circularizing a target nucleic acid molecule using a target-specific probe as a circularization template, or by circularizing a target-specific probe using a target nucleic acid molecule as a circularization template.

[0007] Circular RCA template can be conveniently provided using symmetric selection probes, which include longer nucleic acid chains suspended at the two ends of shorter nucleic acid chains, and the longer chains include sequences complementary to the sequences in the target nucleic acid molecules at their ends, as described in US 7883849. After hybridization, the selector and the target nucleic acid fragment are joined together by connection to obtain a circular nucleic acid molecule. Alternative methods for selecting and amplifying nucleic acids relate to the general cyclization of genomic fragments, as described in Drmanac et al. 2010. Science 327, 78-81 and US 8518640. Methods based on selection probes for selecting target nucleic acid sequences are also provided in WO 03 / 044229. However, it has not been demonstrated that cyclization and amplification RNA templates are in this way.

[0008] In alternative techniques, instead of cyclization target nucleic acid, target nucleic acid can be used as a template for connecting one or more probes. For example, so-called " padlock probe " can be used to detect target nucleic acid sequence, as described in, for example, US 5871921. Padlock probe is a linear nucleic acid molecule, which comprises a sequence complementary to the target nucleic acid molecule at its 3' and 5' ends, so that after the padlock probe hybridizes with its target nucleic acid molecule, the end of the probe is juxtaposed to connect. Such method adaptation leads to the development of the following technology: wherein the end of the probe is combined with a sequence separated by one or more bases in the target nucleic acid molecule, and wherein the gap between each end of the probe uses nucleic acid polymerase (Hardenbol et al. 2003.Nat Biotechnol 21,673-678) or uses connexon (or gap) oligonucleotide (Weibrecht et al. Nature protocols 8,355-372) to fill. Detection technology based on padlock probe has recently been shown to effectively detect pathogens in samples.

[0009] However, detection and amplification methods based on padlock probes typically utilize DNA as a target and DNA probes, because this allows a high level of sequence specificity to be obtained, and allows effective amplification (and detection) of any formed connection products. RNA-templated connections of DNA probes have been demonstrated, and have been demonstrated to be sensitive enough to distinguish SNPs in RNA templates (Nilsson et al. 2001. Nucleic acids research 29, 578-581). Other studies have also demonstrated the use of DNA padlock probes to detect specific miRNAs (Zhao et al. 2015. Acta Biochim Biophys Sin 47, 130-136; Jonstrup et al. 2006. RNA 12, 1747-1752). However, DNA connection assays are typically performed better using DNA templates rather than RNA templates (Nilsson et al. 2000. Nature Biotechnology 18, 791-793), and it has been found that DNA connections using RNA templates are inefficient, and it has also been found that highly variable reaction kinetics are still present even in closely related sequences. Furthermore, most ligases are intolerant to RNA templates, and tolerant ligases have been shown to have poor end-joining fidelity, or in other words, poor ability to distinguish probes that correctly hybridize to their target sequences.Thus, typically, methods in the art generate cDNA molecules from target RNA molecules prior to detection.

[0010] It has been demonstrated that target-dependent ligation of ribonucleotide probes or probes containing ribonucleotides and deoxyribonucleotides can be used for RNA targets (Zhao et al., supra). Specifically, the ligation efficiency of various combinations of 7-mer ribonucleotide or deoxyribonucleotide probes using RNA ligation templates was tested, and it was shown that the ligation of such probes can be templated by RNA templates. However, it has not been demonstrated that amplification of ligation products containing both ribonucleotides and deoxyribonucleotides is poor. In addition, the polymerase typically used in the RCA method has poor processability for templates containing long segments of ribonucleotides.

[0011] Therefore, there remains a need for techniques that allow for the effective detection of a wider range of nucleic acid targets (i.e., including RNA targets), which not only incorporate an effective ligation step, but also allow for amplification of the resulting ligation product to allow for detection of the target. Although probes and methods have been developed primarily for the detection of RNA targets, it has been found that certain novel probes and methods of the present invention also show advantages in the detection of DNA targets, and therefore they may be more widely applied. Summary of the invention

[0012] The method of the present invention uses a probe provided in one or more parts, which is complementary to and hybridizes with a target nucleic acid molecule and is ligated in a target-dependent manner, wherein the probe is primarily composed of DNA but contains ribonucleotides at specific positions relative to the ligation site, which are used to increase the efficiency and fidelity of the ligation templated by the target nucleic acid molecule. Once the ligation product is formed, it is amplified and the resulting amplified product is detected, thereby detecting the target RNA molecule.

[0013] Surprisingly, in the process of preparing the present invention, it is observed that providing ribonucleotides rather than deoxyribonucleotides at or near the ligatable end of the probe enhances the connection efficiency of the probe, and particularly causes the connection efficiency to increase when using RNA to connect the template. This may also be beneficial in the context of detecting DNA targets when detecting RNA and DNA targets together (i.e., in the case of the connection of DNA target templated probes). The ribonucleotide sugar-phosphate backbone in the known oligonucleotides prefers a specific conformation due to the presence of 2'-OH that suppresses the conformation of C2'-in ribose. When binding to the target RNA molecule, the specificity of the ligase to the probe comprising ribonucleotides may also be higher than that to deoxyribonucleotides. Without wishing to be bound by theory, it is believed that compared with the corresponding probe that does not comprise ribonucleotides, the presence of ribonucleotides at or near the ligatable end of the probe allows one or more ends of the probe participating in the templated connection to be recognized and connected by the ligase with higher efficiency and fidelity.

[0014] It was also observed that when the number of consecutive ribonucleotides in the ligation probe consisting mainly of DNA was kept to a minimum, the efficiency with which the ligation product could be amplified was not significantly inhibited. Thus, it was found that it was possible to use a probe comprising one or more ribonucleotides to increase the ligation efficiency while allowing for efficient amplification of the resulting ligation product.

[0015] In its broadest sense, the present invention provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising:

[0016] (a) contacting the sample with a ligatable probe and hybridizing the probe to the target nucleic acid molecule;

[0017] (b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate any probes that have hybridized to the target nucleic acid molecule;

[0018] (c) amplifying the ligation probe from step (b) using a DNA polymerase; and

[0019] (d) detecting the amplification product from step (c), thereby detecting the target nucleic acid sequence;

[0020] wherein each probe is provided in one or more parts, each part having at least one target-specific binding site that is complementary to a cognate probe binding site at or adjacent to the target nucleic acid sequence and hybridizes to the target nucleic acid molecule, such that, optionally following a step of cleaving the hybridization probe and / or extending its 3' end using the target nucleic acid molecule as a template, the ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and

[0021] wherein the probe comprises at least one ribonucleotide at or near the ligation site, optionally after the cleavage and / or extension steps, and the ligation probe consists essentially of DNA and comprises no more than 4 consecutive ribonucleotides.

[0022] The detection method of the present invention can therefore be used to detect a target DNA sequence in a target DNA molecule and / or a target RNA sequence in a target RNA molecule. It is particularly preferred that the target nucleic acid sequence is an RNA sequence and the target nucleic acid molecule is an RNA molecule.

[0023] In one embodiment, the method includes using at least one probe for an RNA target sequence and at least one probe for a DNA target sequence to detect both RNA and DNA target sequences. Therefore, the target RNA and DNA molecules can be in the same sample. In this case, when using RNA ligase, it is beneficial for the DNA targeting probe to also contain at least one ribonucleotide at or near the ligation site when hybridizing with the ligatable ends juxtaposed to ligate.

[0024] The present invention therefore provides a kind of method, it allows to effectively connect the probe that comprises both DNA and RNA nucleotide, and then directly amplify the connection product from the ligation reaction templated by the target nucleic acid molecule, i.e., there is no need to first provide the cDNA molecule complementary to the target molecule. This therefore provides a kind of detection method that is simplified with respect to the methods previously known in the art. In addition, under the background of detecting RNA, because the demand of preparing independent cDNA molecule is omitted, the detection of target RNA sequence in target RNA molecule can be carried out more easily together with detecting other nucleic acid sequences (such as target DNA sequence). This can for example allow to detect target RNA and DNA sequence simultaneously in the same sample.

[0025] A probe for use in such a method is also provided. More particularly, circularizable probes are of particular interest, and in this regard, the present invention therefore provides a chimeric DNA-RNA padlock probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein:

[0026] (i) the probe comprises one or more parts, each part having at least one target-specific binding site, the at least one target-specific binding site being complementary to a cognate probe binding site at or adjacent to the target nucleic acid sequence and hybridizing to the target nucleic acid molecule, such that, optionally after a step of cleaving the hybridization probe and / or extending its 3' end using the target nucleic acid molecule as a template, the ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template to create one or more ligation sites at or adjacent to the target nucleic acid sequence, wherein ligation at the ligation sites circularizes the probe;

[0027] (ii) the probe comprises at least one ribonucleotide at or near the ligation site; and

[0028] (iii) The probe, when ligated to form a circle, consists essentially of DNA and comprises no more than 4 consecutive ribonucleotides.

[0029] Therefore, in a specific embodiment, the present invention provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising:

[0030] a) contacting the sample with a padlock probe and hybridizing the probe to the target nucleic acid molecule;

[0031] b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate and thereby circularize any probes that have hybridized to the target nucleic acid molecule;

[0032] c) amplifying the ligated circularized probe from step (b) by rolling circle amplification with a DNA polymerase; and

[0033] d) detecting the amplification product from step (c), thereby detecting the target nucleic acid sequence;

[0034] wherein each padlock probe is provided in one or more parts, each part having at least one target-specific binding site that is complementary to a cognate probe binding site at or adjacent to the target nucleic acid sequence and hybridizes to the target nucleic acid molecule, such that, optionally after a step of cleaving the hybridization probe and / or extending its 3' end using the target nucleic acid molecule as a template, the ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and

[0035] wherein the probe comprises at least one ribonucleotide at or near the ligation site, optionally after the cleavage and / or extension steps, and the ligated circularized probe consists essentially of DNA and comprises no more than 4 consecutive ribonucleotides.

[0036] The probe of the present invention can be used to detect a target DNA sequence in a target DNA molecule and / or a target RNA sequence in a target RNA molecule, as discussed below. Therefore, in a preferred embodiment, the probe is capable of binding to and detecting a target RNA sequence in a target RNA molecule.

[0037] Since the probe may be provided in one or more parts, there may be more than one ligation junction. In other words, one or more probe parts may each comprise or produce (i.e., by cleavage or extension) ligatable 5' and 3' ends, and the probe as a whole may comprise or produce one or more ligatable 5' ends and one or more ligatable 3' ends.

[0038] In some embodiments, the probe can comprise two or more parts and two or more ligation junctions can be created. A ligation junction can therefore be provided between two different probe parts (or more particularly, between the ligatable ends of two different probe parts, or produced by the ends of two different probe parts), or between the two ends of a single part probe, or produced by the ends of a single part probe.

[0039] After connection, a probe comprising at least one ribonucleotide at or near the connection site is provided. The connection site is the site where the terminal 5' phosphate group at the 5' connectable end of the probe is connected to the terminal 3' hydroxyl group at the 3' connectable end of the probe, and the connection can occur when the respective connectable ends are directly juxtaposed and the respective complementary base pairs in the target nucleic acid molecule are correctly hybridized. Therefore, the connection site is once formed to be the binding point between the nucleotides at the 3' and 5' connectable ends of the probe or probe part, and its position is limited by the phosphodiester bond formed between these nucleotides. Therefore, according to the method and probe of the present invention, at least one nucleotide (i.e., the nucleotides providing the terminal 5' phosphate group or the 3' hydroxyl group) or the nucleotide near the connection site that participates in the ligation reaction is a ribonucleotide. Preferably, at least one nucleotide at or near the connectable 3' end at the connection site is a ribonucleotide. In other words, in a preferred embodiment, when hybridizing with the target nucleic acid molecule so that the 3' and 5' ends are juxtaposed to connect, the probe comprises at or near the connectable 3' end at least one ribonucleotide.

[0040] Therefore, it will be seen that after connection, the probe comprises at least one ribonucleotide at or near the connection site (or more particularly, at or near one or more connection sites), and its presence enhances the specificity and efficiency of connection during the ligation reaction. According to the present invention, in the presence of two or more connection binding sites, at or near one or more of these connection binding sites, at least one ribonucleotide may be present. Not necessary, but preferably, at or near each connection binding site. Therefore, the method of the present invention can be considered to provide a means for enhancing the connection of the primary DNA probe using a template target nucleic acid molecule (such as a target RNA molecule). In other words, the method of the present invention can be used to improve the detection of target nucleic acid molecules in a sample. Significantly, by providing a connection product comprising no more than four continuous ribonucleotides, the method also allows the amplification of the connection product without significant hindrance.

[0041] Any nucleotide at or near the attachment site can be a ribonucleotide rather than a deoxyribonucleotide. The term "near" refers to a position within 5 nucleotides of the attachment site in this context, i.e., 5 nucleotides 3' of the attachment site and 5 nucleotides 5' of the attachment site. Therefore, any ribonucleotide within 5 nucleotides of the attachment site can be a ribonucleotide. Ribonucleotides can preferably be provided in 4 nucleotides of the attachment site, or more preferably in a position within 3 nucleotides of the attachment site. In another embodiment, ribonucleotides can be within 2 nucleotides of the attachment site. Therefore, the terminal nucleotide and / or the penultimate nucleotide of the terminal that can be attached at 3' or 5' can be a ribonucleotide. In some embodiments, the penultimate nucleotide at the 3' or 5' attachment end (preferably 3' can be attached to the end) can be a ribonucleotide.

[0042] In a preferred embodiment, ribonucleotides can be at the 3' or 5' connectable ends of the attachment site. In this article, the term 'at ...' specifically refers to the terminal nucleotide at the end of the oligonucleotide, i.e., the nucleotide of the hydroxyl or phosphate group provided for connection. Therefore, in one embodiment, mentioning that the probe comprises ribonucleotides at the attachment site refers to the terminal 3' nucleotide of the 3' connectable end or the terminal 5' nucleotide of the 5' connectable end being a ribonucleotide. Preferably, the 3' connectable end at the attachment site is a ribonucleotide.

[0043] More than one nucleotide at or near the joining site can be a ribonucleotide. Therefore, in certain embodiments, more than one ribonucleotide at or near the joining site (i.e. any one of the 10 nucleotides within 5 nucleotides of the joining site) can be a combined ribonucleotide, provided that the joining probe comprises no more than 4 continuous ribonucleotides discussed elsewhere.

[0044] In a preferred embodiment, the probe comprises at least one ribonucleotide at the ligatable end of the probe or its portion. Preferably, the probe comprises at least one ribonucleotide at the 3' ligatable end at the connection site. In other words, the nucleotide at the ligatable 3' end of the probe or its portion is preferably a ribonucleotide rather than a deoxyribonucleotide.

[0045] In some embodiments, the probe or probe portion does not include ribonucleotides at or near the 5' connectable end at the connection site, and particularly does not include ribonucleotides at the 5' connectable end at the connection site. In some embodiments, in the case where the 5' connectable end is provided by the 5' end of the probe or probe portion (i.e., in the case where the 5' connectable end is not produced by cutting the hybridization probe), the 5' terminal nucleotide of the probe or probe portion is preferably not a ribonucleotide. In other words, in a preferred embodiment of the present invention, the nucleotide at the connectable 5' end of the probe or its portion is a deoxyribonucleotide. In such embodiments, the 3' hydroxyl group of the nucleotide (it is a ribonucleotide or a deoxyribonucleotide) at the connectable 3' end from the probe or its portion is connected to a 5' deoxyribonucleotide.

[0046] As briefly described above, the above-mentioned ligatable ends that ultimately form the ligation site need to be directly juxtaposed and hybridized with their respective complementary base pairs in the target nucleic acid molecule in order to be connected. However, according to the design of the probe and / or its parts, the ligatable ends can be juxtaposed in a variety of ways to be connected.

[0047] In a specific embodiment, the 3' end of the probe or portion thereof is a ligatable 3' end at the ligation site. Thus, in such an embodiment, the ligatable 3' end is provided at (or alternatively expressed from) the 3' end of the probe, or in other words, the probe hybridizes to the target nucleic acid molecule such that the 3' end of the probe can be ligated to the ligatable 5' end at the ligation site.

[0048] Therefore, in a preferred embodiment, the probe comprises at least one ribonucleotide at or near the 3' end of the probe or its portion (but preferably at the 3' end). Therefore, the probe can not only comprise at least one ribonucleotide at or near the connectable 3' end at the connection site, as described above, but also the 3' end of the probe or its portion itself can be a connectable 3' end as described herein. Therefore, in this embodiment, the probe or probe portion provided in the step (a) of the inventive method can comprise at or near its 3' end at or near at least one ribonucleotide, the end hybridizes with the target nucleic acid sequence and is a connectable 3' end for the connection reaction of step (b). In other words, in such embodiments, the nucleotide at the 3' end of the probe or its portion participates in the connection reaction, and is connected to the connectable 5' end at the connection site during the process of the detection method.

[0049] In another embodiment of the present invention, the 5' end of the probe is a ligatable 5' end at the ligation site. Thus, a ligatable 5' end may be provided at the 5' end of the probe or a portion thereof, or in other words, the probe may hybridize with the target nucleic acid molecule such that the 5' end of the probe or a portion thereof may be ligated to a ligatable 3' end at the ligation site.

[0050] Thus, in a specific embodiment, two ligatable ends may be provided at the ends of the probe or probe portion, or in other words, the probe may hybridize to the target nucleic acid molecule so that the ligatable ends of the probe or probe portion are directly juxtaposed to be connected to each other using the target nucleic acid molecule as a ligation template without any further modification or processing (e.g., cutting or extension) of the probe. In other words, the probe or each portion thereof may include at least one target-specific binding site at one or more of its ends so that each end of the probe (or portion thereof) may hybridize to a directly adjacent homologous probe binding site in the target nucleic acid molecule so that a ligatable end is provided for connection. In other words, in certain embodiments, step (b) may consist of a ligation step without any other step to provide the 5' and 3' ends of the probe or probe portion juxtaposed for connection.

[0051] However, it is not necessary for the probes to contain target-specific binding sites at their respective ends which hybridize to homologous sequences directly adjacent to each other in the target nucleic acid molecule in order to bring the ligatable ends into juxtaposition for ligation as described above, and optionally one or both ligatable ends of the probes may be generated only in one or more processing steps before ligation occurs.

[0052] In a preferred embodiment of the invention, the 5' ligatable end of the probe or part thereof may be created by cleavage when the probe is hybridized to the target nucleic acid molecule (ie by cleavage of the hybridization probe).

[0053] Therefore, the probe may include an additional sequence 5' of the probe or probe portion (or outside) that provides a 5' ligatable end, so that the 5' ligatable end of the probe or probe portion can only be created after the additional sequence has been cut (cut). In other words, the probe or probe portion may include a 5' additional sequence. Therefore, the probe or probe portion may include a first target-specific target binding site located inside the 5' end of the probe or probe portion, so that the probe or probe portion hybridizes with the target nucleic acid molecule and provides a portion that can connect to the 5' end at the connection site (i.e., the target-specific binding site) when appropriate, and includes an additional sequence that does not hybridize to the target nucleic acid molecule at its 5' end. In this way, any additional sequence that has been removed by cutting at the 5' ligatable end of the probe or probe portion can be connected, and probe connection can be performed.

[0054] This additional sequence 5' of the first target-specific binding site which does not hybridize to the target nucleic acid molecule thus forms a 5' flap which can be removed by cleavage, thereby creating a 5' ligatable end of the probe or probe portion.

[0055] It should be understood that in a multi-part probe (i.e., a probe comprising two or more parts), more than one 5' flap can be formed. For example, in a two-part probe (e.g., a padlock probe comprising a backbone oligonucleotide and a gap oligonucleotide, the gap oligonucleotide hybridizing with the target between the two hybridization ends of the backbone oligonucleotide, as further described below), the 5' end of the backbone oligonucleotide can form a 5' flap. In another embodiment, the 5' end of the gap oligonucleotide can form a 5' flap, and in yet another embodiment, the 5' ends of the backbone and the gap oligonucleotide can both form a 5' flap. In addition, the multi-part padlock probe can include more than one gap oligonucleotide. Therefore, two or more gap oligonucleotides can each form a 5' flap.

[0056] Thus, following step (a), the method may include the step of cleaving any probe that has hybridised to the target nucleic acid molecule. This step removes the (or any) 5' flap that is formed when the probe or probe portion hybridises to the target nucleic acid molecule.

[0057] The step of cleaving the hybridization probe to remove the 5' flap can be performed using an enzyme. Any enzyme capable of performing a reaction that removes the 5' flap can be used in this step, i.e. any enzyme capable of cleaving, degrading or digesting the 5' single-stranded sequence that is not hybridized to the target nucleic acid molecule, but typically this will be an enzyme with 5' nuclease and / or structure-specific cleavage activity.

[0058] The 5' flap formed in this way can be recognized by a structure-specific cutting enzyme, such as an enzyme that can recognize the binding point between the single-stranded 5' overhang and the DNA duplex and cut the single-stranded overhang. It should be understood that the branched three-stranded structure as a substrate for the structure-specific cutting enzyme can be formed by the 5' end of one probe part and the 3' end of another probe part when both have hybridized with the target nucleic acid molecule, as well as by the 5' and 3' ends of one probe. Enzymes suitable for such cutting include Flap endonucleases (FENS), which are enzymes with endonuclease activity and can catalyze the hydrolytic cutting of phosphodiester bonds at the binding point of single-stranded and double-stranded DNA. Therefore, in a preferred embodiment, the additional sequence 5' of the first target-specific binding site is cut by a structure-specific cutting enzyme, such as a Flap endonuclease. In a preferred embodiment, the enzyme may be a native or recombinant archaeal FEN1 enzyme from Pyrococcus furiosus (Pfu), Archaeoglobium fulgidus (Afu), Methanococcus jannaschii (Mja) or Methanobacterium thermoautotrophicum (Mth), for example as described in Ma et al. 2000. JBC 275, 24693-24700.

[0059] In other embodiments, enzymes capable of recognizing and degrading single-stranded oligonucleotides with free 5' ends can be used to cut additional sequences (5' flaps) from structures as described above. Therefore, enzymes with 5' nuclease activity can be used to cut 5' additional sequences. This 5' nuclease activity can be a 5' exonuclease and / or a 5' endonuclease activity. 5' nucleases are capable of recognizing the free 5' end of single-stranded oligonucleotides and degrading the single-stranded oligonucleotides. 5' exonucleases degrade single-stranded oligonucleotides with free 5' ends by degrading the oligonucleotides from their 5' ends into constituent mononucleotides. 5' endonuclease activity can cut the 5' flap sequence within one or more nucleotides. In addition, 5' nuclease activity can occur by the enzyme allowing the single-stranded oligonucleotide to span the duplex region after recognizing the free 5' end, and cleaving the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cleaving the entire 5' single-stranded region, such as for Taq DNA polymerase and its 5' nuclease as described in Lyamichev et al. 1999. PNAS 96, 6143-6148. Preferred enzymes with 5' nuclease activity include exonuclease VIII, or natural or recombinant DNA polymerases from Thermus aquaticus (Taq), Thermus thermophilus or Thermus flavus, or nuclease domains therefrom.

[0060] In certain embodiments where the probe or probe portion comprises an additional sequence at its 5' end as described above, one or more nucleotides within the additional sequence may be complementary to homologous nucleotides in the target nucleic acid molecule, but may not hybridize simultaneously with the 3' attachable end of the probe or probe portion (which may be a different probe portion, as mentioned above) to the target nucleic acid molecule. In particular, in this way, one or more nucleotides at the 3' end of the additional sequence (i.e., at the 3' end of the 5' flap) may be complementary to homologous nucleotides in the target nucleic acid molecule. Because they cannot hybridize to the target nucleic acid molecule and are prevented from doing so by the 3' attachable end of the probe or probe portion, the additional sequence may be considered to form a replacement flap, and the nucleotides within the additional sequence that are complementary to homologous nucleotides in the target nucleic acid molecule may be considered to be replacement nucleotides or replacement bases.

[0061] Therefore, in a preferred embodiment, one or more nucleotides at the 3' end of the additional sequence (i.e., at the end of the additional sequence closest to the first target binding site) are complementary to homologous nucleotides in the target nucleic acid molecule, wherein the nucleotides cannot hybridize with the 3' connectable end of the probe or the 3' connectable end of another probe portion to the target nucleic acid molecule at the same time. In other words, the one or more nucleotides at the 3' end of the additional sequence can be prevented from binding to the target nucleic acid molecule by the 3' connectable end, or can be considered to be displaced from the target nucleic acid molecule. Therefore, these nucleotides can be referred to as replacement nucleotides.

[0062] Any such additional sequence needs to be removed by cleavage before probe ligation can occur. Probes with such additional sequence at their (or one) 5' end (i.e. probes in which the first target-specific binding site is located within the 5' end of the probe or probe portion) can therefore be considered to need to be activated (i.e. by cleavage) before ligation.

[0063] Surprisingly, for DNA and RNA target nucleic acid molecules, it has been found that in the 5' additional sequence of the probe, in particular at the 3' end of the 5' additional sequence, there are ribonucleotides (i.e., the 3' nucleotides of the additional sequence) that enhance the removal (i.e., cutting) efficiency of the additional sequence. This effect is particularly noted in the so-called invasive cleavage reaction described in more detail below. The additional sequence described above can therefore comprise one or more ribonucleotides in certain embodiments. In certain embodiments of the present invention, one or more nucleotides at the 3' end of the 5' additional sequence can be ribonucleotides. More particularly, one or more nucleotides at the 3' end of the 5' additional sequence as described above that are complementary to the homologous nucleotides in the target nucleic acid molecule (i.e., the replacement nucleotides as described above) can be ribonucleotides. In a specific embodiment, the additional sequence can be composed entirely of ribonucleotides.

[0064] The enhanced cleavage of the 5' additional sequence of the probe is separate from the enhanced ligation efficiency that results from having ribonucleotides at or near the ligation site in the chimeric DNA-RNA probe. Thus, the effects described above can also be seen in probes that do not contain ribonucleotides at or near the ligation site after cleavage.

[0065] In another aspect, the present invention therefore provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising:

[0066] (a) contacting the sample with a ligatable probe and hybridizing the probe to the target nucleic acid molecule;

[0067] (b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate any probes that have hybridized to the target nucleic acid molecule;

[0068] (c) amplifying the ligation probe from step (b) using a DNA polymerase; and

[0069] (d) detecting the amplification product from step (c), thereby detecting the target nucleic acid sequence;

[0070] wherein each probe is provided in one or more parts, each part having at least one target-specific binding site, the at least one target-specific binding site being complementary to a homologous probe binding site at or adjacent to the target nucleic acid sequence and hybridizing to the target nucleic acid molecule, wherein one target-specific binding site of the probe or at least one target-specific binding site of a probe part is located within the 5' end of the probe or probe part, so that after hybridization of the probe or probe part to the target nucleic acid molecule, the probe or probe part contains an additional sequence 5' to the target-specific binding site, which additional sequence 5' does not hybridize to the target nucleic acid molecule and forms a 5' flap, wherein one or more nucleotides at or near the 3' end of the 5' additional sequence are ribonucleotides, and wherein after a step of cutting the hybridization probe to remove the 5' flap, and optionally after a step of extending its 3' end using the target nucleic acid molecule as a template, the ligatable ends of the probe or probe part are juxtaposed to be ligated to each other using the target nucleic acid molecule as a ligation template, thereby creating a ligation site at or adjacent to the target nucleic acid sequence.

[0071] In particular, the nucleotide at the 3' end of the 5' additional sequence may be a ribonucleotide. Optionally, one or more nucleotides near the 3' end of the 5' additional sequence may also be a ribonucleotide.

[0072] The target nucleic acid sequence can be a target DNA sequence or a target RNA sequence, and the target molecule can be a target DNA molecule or a target RNA molecule. As discussed above, this method of this aspect of the invention can also be used to detect more than one target sequence (included in more than one target molecule), and can include detecting RNA and DNA target sequences in RNA and DNA target molecules (e.g., in the same sample).

[0073] In another aspect, the present invention thus provides a probe for use in such a method. This aspect of the present invention provides a chimeric DNA-RNA probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein:

[0074] (i) the probe comprises one or more parts, each part having at least one target-specific binding site, the at least one target-specific binding site being complementary to a cognate probe binding site at or adjacent to the target nucleic acid sequence and hybridizing to the target nucleic acid molecule, wherein the first target-specific binding site of the probe or the target-specific binding site of the probe part is located within the 5' end of the probe or the probe part, and the probe or the probe part comprises an additional sequence 5' to the target-specific binding site, such that when the probe or the probe part hybridizes to the target nucleic acid molecule, the additional sequence forms a 5' active A flap, the 5' flap does not hybridize to the target nucleic acid molecule and can be removed by cutting to produce a ligatable 5' end that can be connected to the 3' end of the probe or probe portion, wherein the additional sequence contains one or more ribonucleotides (preferably at its 3' end), so that after the step of cutting the hybridization probe or probe portion and optionally after the step of extending its 3' end using the target nucleic acid molecule as a template, the ligatable ends of the probe or probe portion are juxtaposed to be connected to each other using the target nucleic acid molecule as a connection template, thereby creating one or more connection sites at or adjacent to the target nucleic acid sequence.

[0075] In a preferred embodiment, the nucleotide at the 3' end of any additional sequence forming the 5' flap is complementary to a homologous nucleotide in the target nucleic acid molecule that is identical to the nucleotide at the 3' end of the probe. Thus, one or more nucleotides at the 3' end of any additional sequence (i.e., at the end of the additional sequence closest to the first target binding site) can be complementary to a homologous nucleotide in the target nucleic acid molecule, wherein the nucleotide cannot hybridize with the 3' attachable end of the probe or the 3' attachable end of another probe portion simultaneously with the target nucleic acid molecule.

[0076] Such a probe may optionally be a single part or a two (or more) part padlock probe, or a probe comprising two or more parts as defined elsewhere herein, except that it does not comprise a ribonucleotide at or near the site of ligation.

[0077] Probes with 5' additional sequences that need to be removed by cutting before connection are well known in the art, for example in the form of invasion probes for invasion assays. Invasion assays in the art typically include the use of a first probe and a second probe with a target-specific binding site, wherein the first probe comprises a first target binding site located inside the 5' end of the probe. In this assay, the first probe and the second probe are typically designed to be complementary and capable of hybridizing with a homologous binding site in a target nucleic acid molecule, so that the first probe hybridizes with the 5' portion of the target nucleic acid molecule, and the second probe directly hybridizes with the 3' portion of the target nucleic acid molecule in juxtaposition with the first probe, and prevents the nucleotides at the 3' end of the additional sequence of the first probe from hybridizing with the target nucleic acid molecule. In other words, in the invasion assay, the nucleotides at the 3' end of the 5' additional sequence are replacement nucleotides as described above. Since the second probe prevents the 3' end of the additional sequence of the first probe from hybridizing with the target nucleic acid molecule, and subsequently cuts the 5' additional sequence, such methods are also referred to as invasive cleavage reactions.

[0078] Therefore, the probe of the present invention can be an invasion probe. Such probes are particularly useful for detecting single nucleotide polymorphisms. Therefore, the detection method of the present invention can be used to detect single nucleotide polymorphisms or any variant bases in the target nucleic acid sequence. The probe used for this method can be designed to make the 3' connectable end of the probe complementary to the nucleotide (variant nucleotide) of interest in the target molecule and can hybridize with it, and the nucleotide at the 3' end of the 5' additional sequence at the 5' end of the probe or the 5' end of another different probe part is complementary to the same described nucleotide, but is prevented from hybridizing with it (i.e., it is a replacement nucleotide as described above) by the 3' connectable end. The probe is cut to remove the additional sequence to provide a 5' connectable end, if the 3' connectable end is correctly hybridized with the target nucleic acid molecule (i.e., complementary to it), then the 5' connectable end can be connected to the 3' connectable end of the probe or the probe part. The probe designed according to this principle provides a high degree of distinction between the different variants at the position of interest, because only the probe with the complementary nucleotide at the 3' connectable end and the position of interest can participate in the ligation reaction. In one embodiment, the probe is provided with a single part, and 3' and 5' connectable ends are provided by the same probe.

[0079] Recently, it has been demonstrated that alternative designs of invasive probes provide comparable results to the designs outlined above (Krzywkowski et al. 2017. Nucleic Acids Research 45, e161). Although this design is specifically disclosed in invasion assays utilizing lock probes (such as the "invasion lock" (iLock) probes described in detail below), certain features of such probes may be generally applicable to invasive probes.

[0080] In this alternative design, the probe (which is provided as a probe with one part in the context of an iLock probe) is designed such that the 3' nucleotide of the 3' target-specific binding site (such as the second target-specific binding site described herein) in the padlock probe is complementary to and capable of hybridizing with a nucleotide in the target nucleic acid molecule 3' of the nucleotide of interest (i.e., the nucleotide immediately 3' to the variant nucleotide), and the 5' nucleotide of the 5' target-specific binding site (such as the first target-specific binding site described herein) in the padlock probe is complementary to and capable of hybridizing with a nucleotide of interest (i.e., the variant nucleotide) in the target nucleic acid molecule. Such a probe comprises an additional sequence 5' to the first target-specific binding site such that the first target-specific binding site is located internal to the 5' end of the probe. The 3'-most terminal nucleotide of the additional sequence is complementary to a nucleotide in the target nucleic acid molecule 3' of the nucleotide of interest (i.e., 3' immediately adjacent to the nucleotide of interest / variant nucleotide), but is prevented from hybridizing with the target nucleic acid molecule by the 3' connectable end of the probe (i.e., the second target-specific binding site), and is therefore a replacement nucleotide. As above, a similar probe design may be provided wherein the 5' target-specific binding site and the 3' target-specific binding site are provided by different probe portions.

[0081] Therefore, more generally, the probe can be designed so that the 3' connectable end of the probe or probe portion is complementary to the nucleotide in the target molecule 3' of the nucleotide of interest (variant nucleotide) and can hybridize with it, and the nucleotide at the 3' end of the 5' additional sequence of the probe or the 5' end of another different probe portion is complementary to the nucleotide 3' of the nucleotide of interest (the nucleotide of the 3' next to the variant nucleotide), but is prevented from hybridizing with it by the 3' connectable end of the probe or probe portion. Cutting to remove the additional sequence provides a 5' connectable end, if the 5' connectable end is correctly hybridized with the target nucleic acid molecule (i.e. complementary to it), then the 5' connectable end can be connected to the 3' connectable end of the probe or probe portion. The probe designed according to this principle provides a high degree of distinction between different variants at the position of interest, because only the probe with the 5' connectable end complementary to the nucleotide at the position of interest can participate in the ligation reaction. As described above, in one embodiment, the probe is provided in a single part, and 3' and 5' connectable ends are provided by the same probe.

[0082] Therefore, in certain embodiments, the method of the present invention can be used to detect variant bases in target nucleic acid molecules. In particular, probes according to any of the "invader" designs mentioned above can be particularly used to detect variant bases in target nucleic acid molecules.

[0083] In such invasive probes that create a 5' flap, it is preferred in some embodiments that, as mentioned above, one or more nucleotides at or near the 3' end of the additional sequence (creating the 5' flap) are ribonucleotides. More particularly, the 3'-most nucleotide or at least the 3'-most nucleotide of the additional sequence (or in other words, the 3' terminal nucleotide of the excised 5' flap) is a ribonucleotide.

[0084] The invasive probe may be provided as a padlock probe, which may be composed of one or more parts, as further described below. Thus, the padlock probe may be a single part padlock that hybridizes to the target nucleic acid molecule to form an "invasive" structure, or it may comprise two or more parts that hybridize to form one or more "invasive" structures having a 5' flap for cleavage.

[0085] In a specific representative embodiment, the probe used in this method is a padlock probe provided as a single circularizable oligonucleotide comprising a first target-specific binding site located within the 5' end of the probe and a second target-specific binding site located at the 3' end of the probe, such that after hybridization of the probe to the target nucleic acid molecule, the probe comprises an additional sequence 5' of the first target binding site that does not hybridize to the target nucleic acid molecule, wherein the nucleotides at the 3' end of the probe are ribonucleotides and wherein the nucleotides at the 3' end of the probe and the 3' end of the additional sequence are complementary to the same nucleotide, wherein when When the nucleotides at the 3' end of the probe and the nucleotides at the 3' end of the additional sequence are complementary to the variant base, the nucleotides at the 3' end of the additional sequence cannot hybridize with the target nucleic acid molecule at the same time as the 3' end of the probe and the additional sequence is removed by cutting to produce a 5' connectable end of the probe, and wherein when the nucleotides at the 3' end of the additional sequence and the nucleotides at the 3' end of the probe are not complementary to the variant base, the nucleotides at the 3' end of the additional sequence are removed by cutting and the nucleotides at the 3' end of the probe do not hybridize with the target nucleic acid molecule, thereby preventing connection.

[0086] In another specific representative embodiment, the probe is a single circularizable oligonucleotide comprising a first target-specific binding site located within the 5' end of the probe and a second target-specific binding site located at the 3' end of the probe, such that after hybridization of the probe to the target nucleic acid molecule, the probe comprises an additional sequence 5' of the first target binding site that does not hybridize to the target nucleic acid molecule, wherein the nucleotides at the 3' end of the probe are ribonucleotides, and wherein the nucleotides at the 3' end of the probe and the 3' end of the additional sequence are complementary to the same nucleotide, wherein the nucleotides at the 3' end of the probe and the 3' end of the additional sequence are complementary to the same nucleotide. The nucleotide at the '-end of the probe is complementary to the nucleotide at the 3' position of the variant base and wherein the nucleotide at the 3'-end of the additional sequence cannot hybridize with the 3'-end of the probe and the target nucleic acid molecule at the same time, wherein when the nucleotide at the 5'-end of the first target-specific binding site is complementary to the variant base, the additional sequence is removed by cutting to produce the 5'-ligatable end of the probe, and wherein when the nucleotide at the 5'-end of the first target-specific binding site is not complementary to the variant base, the nucleotide is also removed by cutting to produce a gap between the 5'-ligatable end and the 3'-ligatable end and prevent ligation.

[0087] In other embodiments, such invasive padlock probes may similarly be provided in two or more parts, for example comprising a backbone oligonucleotide and one or more gap oligonucleotides, as further described below.

[0088] In some embodiments, the probe can be designed to bind two or more non-continuous sequences in the target nucleic acid molecule. In other words, the probe or two probe parts can hybridize with the target nucleic acid molecule with a gap between the corresponding target binding sites of the probe or probe parts. When the end of the probe or probe part has hybridized to leave a gap, the target nucleic acid molecule can be used as an extension template to fill this gap by extending the hybridization 3' end of the probe before being connected to the 5' end. After the 3' end of the probe has been extended to be adjacent to the 5' connectable end, the two ends can be connected by ligation. In this way, the connectable 3' end of the probe can be produced by extension.

[0089] This extended gap-filling embodiment can be particularly useful for detecting highly variable target nucleic acid sequences, since the complement of the target nucleic acid sequence is incorporated into the ligation probe. Thus, after amplification, detection of the amplified ligation probe (e.g., by sequencing) allows the sequence of the target nucleic acid sequence to be determined. In other embodiments, this gap-filling by extension can be combined with the feature of generating a ligatable 5' end by cutting the 5' flap. Thus, in the context of invasive probes, gap-filling extensions can also be used to generate or provide 3' ligatable ends.

[0090] ' Extension gap filling ' embodiment described above needs nucleotide to be incorporated in the extension product, and therefore in this type of embodiment, the mixture of nucleoside triphosphates is needed in the reaction mixture to extend.In some embodiments, the one or more nucleoside triphosphates (one, two, three or all four nucleoside triphosphates) (nATP, nGTP, nCTP, dTTP / rUTP) provided can be ribonucleoside triphosphates.This can allow at least one ribonucleotide to be incorporated into 3 ' can be connected to the end in the process of 3 ' end of the extension probe.Therefore, in one embodiment, three of the nucleoside triphosphate molecules provided can be deoxyribonucleoside triphosphates, and the 4th nucleoside triphosphate molecule can be ribonucleoside triphosphates.Preferably, based on the understanding of the target nucleic acid molecule sequence, the one or more nucleoside triphosphates provided with ribonucleoside triphosphates can be selected to make the 3 ' end of the obtained extension product after polymerization or nearby nucleotide (for example 3 ' at the connection site can be connected to the 3 ' terminal nucleotide of the end) be a ribonucleotide. By way of representative example, if the nucleotide at the 5' end of the gap between the two portions of the probe is known to be a "G", a mixture of nucleoside triphosphates including rCTP can be used to provide an rCTP nucleotide at the 3' ligatable end of the extended probe.

[0091] In some embodiments where the probe or part thereof is extended to provide a 3' connectable end, the probe (or part) provided and hybridized with the target nucleic acid molecule can be composed entirely of DNA, and ribonucleotides can be introduced by extension as described above. Therefore, in this embodiment, a DNA probe can be provided and hybridized with the target molecule, and the connectable probe hybridized with the target nucleic acid molecule can be transformed into a chimeric DNA-RNA probe by an extension step. In other words, in the method of the present invention, a connectable probe hybridized with a target molecule having a connectable 5' end and a 3' end juxtaposed to connect (i.e., ready to connect) can be a chimeric DNA-RNA probe.

[0092] In embodiments where ribonucleotides are introduced into the ligatable probe by an extension reaction, the ribonucleotide can be introduced at or near the ligatable 3' end, for example, the end and / or the penultimate 3' nucleotide of the extended sequence can be a ribonucleotide, and / or the extension reaction can introduce 5 or fewer nucleotides, or no more than 4 nucleotides.

[0093] Any convenient polymerase, preferably a non-displacing polymerase, that can use RNA as an extension template can be used to perform the 'gap filling' extension described above, including RNA polymerases and DNA polymerases. Thus, RNA-dependent RNA polymerases (RdRPs), and / or RNA-dependent DNA polymerases (RdDPs) (e.g., reverse transcriptases) can be used in the 'gap filling' embodiments described above.

[0094] In a preferred embodiment, the polymerase may be able to incorporate both DNA and RNA residues into extension products using an RNA template, such as mutated human mitochondrial DNA polymerase γ comprising an E895A or E895G substitution, which has surprisingly been found to incorporate both rNTP and dNTP nucleoside triphosphates into extension products (Kasiviswanathan et al. 2011. JBC 286, 31490-31500).

[0095] The probe used in the detection method disclosed herein can be provided in one or more parts, each of which contains at least one target-specific binding site, and the at least one target-specific binding site is complementary to the target nucleic acid sequence in the target nucleic acid molecule or to the homologous probe binding site adjacent to the target nucleic acid sequence. Therefore, the probe (or each part thereof) is capable of hybridizing with the target nucleic acid molecule. The probe or probe part hybridizes with the target nucleic acid molecule in the following manner: using the target nucleic acid molecule as a connection template, so that its ends are juxtaposed to connect to each other, optionally after the step of cutting the hybridization probe and / or using the target nucleic acid molecule as a template to extend its 3' end. Therefore, the connection reaction of the step (b) of the detection method of the present invention includes forming at least one phosphodiester bond between the corresponding connectable 3' and 5' ends of a part of the adjacent probe or the probe hybridized with the target nucleic acid molecule.

[0096] In some embodiments, the probe can be provided in two or more parts, each of which has at least one target-specific binding site complementary to the homologous probe binding site in the target nucleic acid molecule. Therefore, in one embodiment, the probe can be provided in two parts, each of which contains a target binding site, and the connection in step (b) includes connecting the 3' connectable end of one part of the probe to the 5' connectable end of another part. As described in more detail above, each connectable end of the probe can be produced by cutting and / or extending before connecting.

[0097] In other embodiments of the present invention, probe can be provided with two or more parts that create two or more connection junctions, for example two, three, four, five or more parts. Each part of the probe can provide (i.e. form or include) 3' and / or 5' connectable ends, and the 3' and / or 5' connectable ends can be juxtaposed to connect and connect in the step (b) of the detection method of the present invention as outlined above. In some embodiments, step (b) therefore can include two or more independent ligation reactions, i.e. the ligation reactions between the adjacent connectable ends provided by two or more parts of the probe.

[0098] Thus, the probe may be provided in more than two parts (e.g., in three parts), each of which has at least one target-specific binding site that is complementary to a cognate probe binding site in a target nucleic acid molecule. In this embodiment, the ligation in step (b) comprises ligation of the 5' ligatable end of the first part of the probe to the 3' ligatable end of the second part of the probe, and ligation of the 5' ligatable end of the second part of the probe to the 3' ligatable end of the third part of the probe.

[0099] In a specific embodiment, one or more parts of the probe provided with more than two parts can be the oligonucleotide for connecting sequencing.In the context of the present invention, by connecting sequencing, allow to detect the target nucleic acid molecule with the unknown sequence (for example target nucleic acid sequence) flanked by known sequences at least one end, thereby allow to determine the unknown sequence.One or more parts of probe as described herein can have the target specific binding site complementary to the homologous probe specific binding site in the target nucleic acid molecule, and can further comprise the nucleotide complementary to the specific nucleotide in the unknown sequence at its 5 ' and / or 3 ' end.The part of probe can only be connected when correctly hybridizing with the nucleotide in the unknown sequence, and therefore the subsequent detection of the connection product of amplification can allow to determine the target nucleic acid sequence.In a specific embodiment, under the background of multi-part lock probe, one or more gap oligonucleotides (as further discussed below) can be the oligonucleotide for connecting sequencing.

[0100] In certain embodiments, and as briefly discussed above, the probe used in the detection method of the present invention can be a padlock probe comprising one or more parts, and the one or more parts are optionally connected together to form a circle after the cutting step. In a typical embodiment, the padlock probe can include a linear single-stranded oligonucleotide (backbone oligonucleotide), which includes two target-specific binding sites, and the two target-specific binding sites are complementary to the homologous probe binding sites in the target nucleic acid molecule, and the target nucleic acid molecule is connected by a sequence (backbone or joint) that is not complementary to the target nucleic acid, and therefore (optionally after cutting and / or extension as discussed above) 3' and 5' can be connected to the ligation reaction. The 5' and 3' ends of the padlock probe can therefore participate in one or more ligation reactions using the target nucleic acid molecule as a ligation template, thereby forming a circular nucleic acid molecule.

[0101] In certain embodiments, a padlock probe can be provided as a single circularizable oligonucleotide comprising a first target-specific binding site at or within the 5' end of the probe and a second target-specific binding site at the 3' end of the probe, wherein the first target-specific binding site and the second target-specific binding site form or constitute the 5' ligatable end and the 3' ligatable end of the probe, respectively.

[0102] In such embodiments, the ligation can be direct, i.e., the 5' and 3' ends of the padlock probe can hybridize to the target nucleic acid molecule directly adjacent to each other, such that the 3' end of the padlock probe is connected to the 5' ligatable end of the padlock probe, thereby forming a circular ligation product. Alternatively, in such embodiments, the 5' and 3' ends of the padlock probe can hybridize to the target nucleic acid molecule with a gap between its target-specific binding sites, and the 3' end of the probe can be extended as described elsewhere herein to provide the 3' ligatable end of the probe for connection to the 5' ligatable end of the padlock probe.

[0103] The padlock probe provided as a single circularizable oligonucleotide may optionally include an additional sequence 5' to its 5' target binding site, which may be cleaved to provide a 5' ligatable end at the ligation site. Thus, in a specific embodiment, the probe may be a single circularizable oligonucleotide comprising a first target-specific target binding site located within the 5' end of the probe and a second target-specific binding site located at the 3' end of the probe, such that after hybridization of the probe to the target nucleic acid molecule, the probe comprises an additional sequence 5' to the first target binding site that does not hybridize to the target nucleic acid molecule, wherein the nucleotides at the 3' end of the additional sequence are ribonucleotides that are complementary to homologous nucleotides in the target nucleic acid molecule but cannot hybridize to the target nucleic acid molecule simultaneously with the 3' ligatable end of the probe, wherein the additional sequence is removed by cleavage to create the 5' ligatable end of the probe.

[0104] The padlock probe can be provided with two or more parts alternatively, i.e., provided as two or more oligonucleotides, which are connected to form a circular oligonucleotide. The first oligonucleotide can be included in the first target specific binding site at its 5' end or inside and the second target specific binding site at its 3' end, and can be hybridized with the target nucleic acid molecule, with a gap between its target specific binding sites. However, rather than extending the 3' end of the padlock probe to provide a 3' connectable end for connection, the second oligonucleotide (and the optional third, fourth etc. oligonucleotides) can be provided, which comprises one or more target specific binding sites complementary to the homologous probe binding site between the first target specific binding site and the second target specific binding site in the target nucleic acid molecule. In this embodiment, the cyclization of the padlock probe includes the 5' and 3' connectable ends of the first oligonucleotide being connected to the 3' and 5' connectable ends of these oligonucleotides (for example, being respectively connected to the 3' and 5' connectable ends of the second oligonucleotide). In such embodiments, the second oligonucleotide and subsequent oligonucleotide can be considered to be gap oligonucleotides. A padlock probe provided in two or more parts may thus be considered to comprise a backbone oligonucleotide (ie the oligonucleotide comprising the first target-specific binding site and the second target-specific binding site) and one or more gap oligonucleotides.

[0105] One or more gap oligonucleotides can be used for sequencing by ligation, as described above.

[0106] In certain embodiments, the padlock probe (or more particularly, the backbone oligonucleotide of the padlock probe) itself can be provided in two or more parts, preferably in two parts, which can be connected at a position different from the connection site formed between the parts of the probe or probe part that hybridizes to the target nucleic acid molecule during the circularization of the padlock probe. In other words, the backbone oligonucleotide of the padlock probe can be provided in two or more parts, which can be connected at a position within a sequence that is not complementary to the target nucleic acid (i.e., a backbone or linker as described above). An example of a padlock probe in which the backbone oligonucleotide is provided in two parts is shown in Fig.17 middle.

[0107] This connection of the backbone oligonucleotide provided in two or more parts can be templated by a connection template, which can hybridize with each of the two or more parts of the backbone oligonucleotide, so that the respective 5' and 3' ends are juxtaposed to connect. The connection template therefore comprises a region complementary to the sequence at the end of each of the two or more parts of the backbone oligonucleotide.

[0108] The ligation template can be a separate oligonucleotide, such as an oligonucleotide that is added to the sample together with the probe or separately, or pre-hybridized with the probe, or can be another target nucleic acid molecule, or another part of the same target nucleic acid molecule (i.e., another separate target sequence at a different position within the same target nucleic acid molecule). Therefore, the ligation template can be a synthetic or natural oligonucleotide, and can be an RNA molecule or a DNA molecule.

[0109] The connection of the part of the backbone oligonucleotide can be direct or indirect, as defined elsewhere herein, or may require gap filling extension or other oligonucleotides (i.e. similar to gap oligonucleotides) hybridized with the connection template between the ends of the two parts of the backbone oligonucleotide. In addition, the connection may require cutting of 5' additional sequences, optionally wherein the nucleotides at the 3' ends of the additional sequences are ribonucleotides, and / or wherein ribonucleotides are provided at or near the connection site, as described elsewhere herein. It can be seen that this cyclization method (wherein the backbone oligonucleotide is provided with 2 or more parts) can be conveniently used for introducing ribonucleotides into the backbone oligonucleotide (e.g., by using rNTP extension) at a site different from the (or one) connection site on the (main) target molecule as a template. The presence of one or more such ribonucleotides at one or more different sites in the backbone oligonucleotide may be beneficial to the (cyclized) padlock probe connected, because this works to accelerate the RCA reaction, as discussed below.

[0110] In the case where more than one connection occurs during step (b) (i.e., in the case where two or more connection sites are formed), any connection site may include at least one ribonucleotide as discussed in more detail above (i.e., the probe may include a ribonucleotide at or near any connection site). Therefore, in the case where there are two or more connection sites, one or more of the connection sites may include at least one ribonucleotide as defined herein. In a specific embodiment of the present invention, the probe may include a ribonucleotide at or near each connection site. Therefore, in a preferred aspect, any one or all of the probes provided in two or more parts may include at least one ribonucleotide at or near the connectable 3' end at the connection site, and in a specific embodiment, each part of the probe may include at or near its connectable 3' end at or near the connection site. More preferably, each part of the probe may include at or near its 3' end. However, it should be noted that in the case where the probe is provided in two or more parts, it is sufficient for only one of the two or more parts to include at or near its connectable 3' end or at or near its 3' end.

[0111] In the case where the probe is provided in two or more parts as described above, the 5' ligatable end of any or all parts of the probe can be created by cutting, as described elsewhere herein. In particular, the target-specific binding site of any or all parts of the probe provided in two or more parts can be located inside the 5' end of the probe part (i.e., an additional sequence 5' of the target binding site that does not hybridize with the target nucleic acid molecule can be included), and the additional sequence can be removed by cutting before connection. Similarly, any two or more parts of the probe can hybridize with non-continuous sequences in the target nucleic acid molecule, and the gap between them can be filled by extending the 3' end of any or all probes as needed, as discussed above.

[0112] The ribonucleotide quantity contained in the probe is not critical, as long as the probe meets the requirement that the connection probe comprises no more than 4 continuous ribonucleotides.More particularly, the connection probe can comprise no more than 3 continuous ribonucleotides, and in a preferred embodiment, no more than 2 continuous ribonucleotides (such as 1 or 2 ribonucleotides).Because the probe can comprise more than one connection site, and ribonucleotides can be present at or near each or multiple connection sites, it should be understood that ribonucleotides can be present at multiple (i.e. two or more) sites in the connection probe, but at each site, there are no more than 4 (or 3 or 2) continuous ribonucleotides.In other words, at each connection site, there can be 1 ribonucleotide or no more than 4 (or 3 or 2) continuous ribonucleotides, in fact, within the scope of the above description, other sites of the whole probe (i.e. not only at the connection site) include ribonucleotides without restriction.Surprisingly, it has been found that there is ribonucleotide in the circular connection probe to allow rolling circle amplification to be carried out faster. Probes for use in the methods of the invention may therefore optionally comprise one or more (and up to 4 consecutive) ribonucleotides at one or more sites (including at one or more positions other than at or near the site of ligation).

[0113] As described above, the probe or probe portion may have an additional sequence 5' of the first target binding site, and this additional sequence forms a removable 5' flap. As described above, the additional sequence (5' flap sequence) may contain one or more ribonucleotides that may be consecutive, and the number is not limited; these ribonucleotides can be removed by cleavage, so that when the probe is connected, no more than 4 (or no more than 3 or 2) consecutive ribonucleotides remain in the probe.

[0114] In some embodiments where the probe is a padlock probe, the backbone oligonucleotide can be made up of DNA completely, and ribonucleotides can be included in one or more gap oligonucleotides (more specifically, at or near its 3' connectable end), and / or they can be introduced by extension, as discussed above. In other embodiments, within the scope of the above, the backbone oligonucleotide can contain one or more ribonucleotides, and for example, a single ribonucleotide can be scattered everywhere. In another embodiment, the backbone oligonucleotide can comprise at least one ribonucleotide at or near the 3' connectable end. In this embodiment, one or more gap oligonucleotides can also contain at least one ribonucleotide at or near its 3' connectable end. Alternatively, ribonucleotides can be provided only in the backbone oligonucleotide. Any combination of these features can be used.

[0115] The present invention may therefore alternatively be viewed as providing a method for detecting a target nucleic acid sequence in a target nucleic acid molecule hybridised to a ligatable chimeric DNA-RNA probe, the method comprising:

[0116] i) ligating a chimeric DNA-RNA probe hybridized to a target nucleic acid molecule;

[0117] ii) amplifying the ligation probe from step (i) using a DNA polymerase; and

[0118] iii) detecting the amplification product from step (ii), thereby detecting the target nucleic acid sequence;

[0119] wherein the ligatable chimeric DNA-RNA probe is provided in one or more parts, each part having at least one target-specific binding site that is complementary to a cognate probe binding site at or adjacent to the target nucleic acid sequence and hybridizes to the target nucleic acid molecule such that the ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and

[0120] wherein the ligatable chimeric DNA-RNA probe comprises at least one ribonucleotide at or near the ligation site, and the ligation probe consists essentially of DNA and comprises no more than 4 consecutive ribonucleotides.

[0121] It will be apparent that according to this method, for example after a cleavage and / or extension step, ligatable ends of probes or probe portions may have been generated which are juxtaposed to each other for ligation, as described in more detail above.

[0122] The target nucleic acid molecule may be any sequence desired for detection, analysis or amplification. Thus, it may be DNA or RNA or a modified variant thereof. Thus, the nucleic acid may be composed of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotides capable of participating in Watson-Crick type or similar base pair interactions. Thus, the nucleic acid may be or may comprise, for example, bisulfite-converted DNA, LNA, PNA or any other derivative containing a non-nucleotide backbone.

[0123] The target nucleic acid molecule may thus be coding or non-coding DNA, such as genomic DNA or a sub-portion thereof, or may be derived from genomic DNA, such as a copy or an amplicon thereof, or it may be cDNA or a sub-portion thereof, or an amplicon or a copy thereof, etc.

[0124] As mentioned above, the target nucleic acid molecule can be a target RNA molecule. The target nucleic acid molecule can be, for example, an RNA molecule or other nucleic acid molecule or nucleotide sequence in an RNA pool, such as humans or from any source, from a transcriptome, or any other nucleic acid (such as an organelle nucleic acid, i.e., mitochondrial or plastid nucleic acid), a naturally occurring or synthetic nucleic acid. The target RNA molecule can therefore be or can be derived from a coding (i.e., pre-mRNA or mRNA) or non-coding RNA sequence (such as tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA). The target RNA molecule can typically be an RNA molecule that is desired to be detected in a sample, in the sense that it is a target of determination, i.e., an analyte. In a preferred embodiment, the target nucleic acid molecule is a microRNA (miRNA). In another preferred embodiment, the target RNA molecule is a 16S RNA, preferably wherein the 16S RNA is from a microorganism (e.g., a pathogenic microorganism) in a sample and can be identified therefrom. Alternatively, the target RNA molecule can be a genomic RNA of a virus with RNA as its genetic material, such as ssRNA or dsRNA. Notably, such viruses include Ebola, HIV, SARS, influenza, hepatitis C, West Nile fever, polio, and measles. Thus, the target RNA molecule can be a sense RNA, antisense RNA, or double-stranded RNA from a viral genome, or a sense RNA from a retroviral RNA genome.

[0125] The method of the present invention has special advantages for detecting short target nucleic acid molecules, and in particular short target RNA molecules, which are usually difficult to amplify and characterize by conventional techniques known in the art. Therefore, in a specific embodiment of the present invention, the length of the target nucleic acid molecule can be less than or at most 100 nucleotides, or more preferably less than or at most 90, 80, 70, 60, 50, 40, 30 or 25 nucleotides in length. Particularly preferred such short nucleic acid molecules are miRNA molecules, and their length is typically 19-25 nucleotides. WO 2015 / 071445 shows a padlock probe with a pair of target-specific binding sites comprising 6 nucleotides, and the target-specific binding sites can each be combined with target nucleic acid molecules and connected in a target-dependent manner. Therefore, shorter target (e.g., RNA) molecules, for example, target molecules comprising at least 18, 17, 16, 15, 14, 13 or 12 nucleotides can also be detected in the method of the present invention. The target nucleic acid molecule can have a plurality of nucleotides in the range between the above-mentioned arbitrary integers.

[0126] The target nucleic acid molecule can be present in the sample. The sample can be any sample containing any amount of nucleic acid, which comes from any source or belongs to any source, and in the sample, the target nucleic acid sequence in the target nucleic acid molecule needs to be detected. Therefore, the sample can be any clinical or non-clinical sample, and can be any biological, clinical or environmental sample in which the target nucleic acid molecule may be present.

[0127] The sample can be any sample containing the target nucleic acid molecule, and includes natural and synthetic samples, i.e., naturally occurring materials or prepared preparations. Naturally occurring samples can be treated or processed before being subjected to the method of the present invention. Include all biological and clinical samples, such as any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, and samples such as cell cultures, cell preparations, cell lysates, etc. Also include environmental samples, such as soil and water samples or food samples. Samples can be freshly prepared, or they can be pre-treated in any convenient manner, such as stored.

[0128] Representative samples therefore include any material that can contain target nucleic acid molecules, including, for example, food and related products, clinical and environmental samples. Sample can be a biological sample, which can contain any virus or cell material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. This biological material can therefore include all types of mammals and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa, etc., or viruses. Representative samples therefore include whole blood and blood-derived products, such as blood plasma, serum and buffy coat, hemocytes, urine, feces, cerebrospinal fluid or other samples of any other body fluid (for example, respiratory secretions, saliva, milk, etc.), tissue, biopsy, cell culture, cell suspension, conditioned medium or cell culture components, etc. Samples can be pre-treated in any convenient or desired manner to prepare for method and purposes of the present invention, for example, by cell lysis or purification, separation of nucleic acid or RNA, etc.

[0129] In a specific embodiment, the sample comprises a microbial cell or virus that has been separated from a clinical sample or a culture of a clinical sample. In such a sample, the target nucleic acid molecule can be a nucleotide sequence present in a microbial cell, such as a nucleotide sequence that can be characterized, differentiated, or identified from a microbial cell or virus at any level, such as at a type, group, class, genus, species, or strain level.

[0130] Probe as described herein, and in fact one or more parts of probe can comprise one or more other sequences, and described other sequences can be used for introducing sequence into connection product, such as label or detection sequence, such as bar code or can distinguish motif, or detect the binding site of probe or primer.Such other sequences can be found at 3' or 5' end (preferably the opposite end of the connectable end of probe or probe part) of probe or probe part, or can be found in the middle of probe or probe part end, such as in a part of the cyclizable main chain oligonucleotide that is not hybridized with target nucleic acid molecule.Labels such as bar codes or probe / primer binding sites can be designed with different needs / purposes, such as, in order to introduce universal or common sequences, so that different connection probes in multiple environments can be processed together, such as to introduce the binding site of universal or common amplification primers.This will enable different connection probes to be amplified together, such as by PCR or RCA as library amplification. Alternatively or in addition, the tag / barcode sequence can be used to "label" different amplified ligation probes so that they can be easily distinguished from each other (i.e., "target" tags or markers), or to label different samples, etc. so that they can be pooled before a common / universal amplification step (i.e., "sample" tags or markers). Thus, in a multiplexed environment, different probes (i.e., probes for different target nucleic acid molecules) can be provided with different tag sequences (e.g., different markers or detection sequences) and / or they can be provided with the same one or more tag sequences, for example, for the introduction of a common or universal sequence. Such methods can preferably be used in combination with sequencing by hybridization, sequencing by ligation, or other next-generation sequencing chemistries, for example, in multiplexed detection of multiple target nucleic acids in a sample.

[0131] The term "detection" is used extensively herein to include any means for determining the presence of a target nucleic acid sequence in a target nucleic acid molecule. It should be understood that in the method of the present invention, the target nucleic acid is detected by detecting the ligation probe (i.e., presence or absence) or any measurement form of the ligation probe amplified in the sample. Therefore, the amplification product of step (c) can be detected as a "reporter" of the target nucleic acid sequence. Therefore, in step (d), detecting the amplification product can include determining, measuring, evaluating or measuring the presence or absence or quantity or position of the ligation probe amplified in any way. The presence of the ligation probe amplified in the sample (i.e., confirming its presence or amount or connection) indicates or identifies the presence of the target nucleic acid sequence, because the successful connection of the probe (allowing amplification to occur) depends on the target nucleic acid molecule, and more particularly the presence of the target nucleic acid molecule. Therefore, detecting the ligation probe amplified allows the determination of the presence of the target nucleic acid sequence.

[0132] Including quantitative and qualitative determination, measurement or assessment, including semi-quantitative. Such determination, measurement or assessment can be relative (for example, when detecting two or more different target nucleic acid molecules in a sample), or absolute. Therefore, the term "quantitative" can refer to absolute or relative quantification when used in the context of one or more target nucleic acid molecules in a quantitative sample. Absolute quantification can be achieved by including one or more control RNA molecules of one or more known concentrations and / or with a known control RNA molecule with reference to the detection level of the target nucleic acid molecule (for example, by generating a standard curve). Alternatively, relative quantification can be completed by comparing the detection level or amount between two or more different target nucleic acid molecules to provide the relative quantification (i.e. relative to each other) of each of two or more different RNA molecules.

[0133] The sequence of the probe can be selected relative to the sequence of the probe and its target nucleic acid molecule. Therefore, although the target complementary region is selected relative to the target nucleic acid molecule to which they are combined, the sequence of the probe remainder is not critical. However, the sequence should be selected to avoid the occurrence of intramolecular hybridization. After selecting or identifying the sequence, any convenient method synthetic probe can be used.

[0134] As used herein, the term "hybridization" or "hybridization" refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex via Watson-Crick base pairing. When two nucleotide sequences share base pair organizational homology, the molecules are "complementary". Therefore, the complementary region in the probe region refers to a portion of the region that is capable of forming a duplex (or in other words, binding to its homologous complementary sequence). These terms are also used to refer to base pair interactions similar to Watson-Crick base pairing, including Hoogsteen base pairing, which is a rarely observed base pairing variation that also allows the third strand to wrap around a double helix assembled in a Watson-Crick pattern to form a triplex.

[0135] The amount of probe added to the sample can be selected to provide a sufficiently low probe concentration in the reaction mixture to minimize non-target specific interactions, i.e., to ensure that the probe does not randomly bind to non-target nucleic acid molecules in the sample to any large or significant extent. However, in general, the probe will be used in an amount that exceeds the target molecule. In representative embodiments, after combining with the sample, the concentration of each probe in the reaction mixture is in the range of about 1 fM to 1 μM, such as 10, about 1 pM to about 1 nM, including about 1 pM to about 100 nM, for example 1, 2, 5, 10, 20, 50 nM.

[0136] Multiple different probes can be added to the sample for multiple determination (e.g., when there are multiple different target nucleic acid molecules in the sample) to detect multiple target nucleic acid molecules in parallel. Multiple determination may involve the detection of tens, hundreds, thousands or even tens of thousands of nucleic acid molecules in the sample. Therefore, multiple determination can include at least 2 different probes, i.e., probes that can hybridize with different first RCA products (directly or indirectly) and thus, for example, detect different analytes. For example, multiple determination can utilize at least 20, 3, 4, 5, 10, 20, 30, 40 or 50 probes, such as 100, 200, 500, 1000, 10000 or more probes. In addition, the detection method of the present invention (i.e., the detection method for detecting target nucleic acid molecules) can be used in combination with a wider method for detecting other nucleic acid molecules in the sample. In a specific embodiment in which the target nucleic acid molecule is a target RNA molecule, this can allow for detection of target RNA molecules in combination with a wider method for detecting other (e.g., non-RNA) nucleic acid molecules in the sample.

[0137] After combining a sample containing a target nucleic acid molecule and one or more probes, the reaction mixture can be incubated for a period of time sufficient to allow the one or more probes to bind to their target nucleic acid molecules in the sample. As described above, after the probes have bound to the target nucleic acid molecules, the probes are connected (optionally after a step of cutting and / or extending the hybridization probes) to produce a connection product that can then be amplified, and the amplified product is detected to detect the target nucleic acid sequence.

[0138] In some representative embodiments, for example, in other determinations of in-situ determination or wherein fixing target nucleic acid molecules, a washing step can be included between adding probe and connecting and / or amplifying connection products. In other words, the target nucleic acid molecules can be captured or fixed on a solid support or substrate, which is washed to remove unbound or non-specifically bound probes. In some embodiments, a washing step can be included between connecting probe and amplifying connection products to remove unconnected probes. In other representative embodiments, a washing step can be included before connecting.

[0139] Where the probe is a multi-part padlock, the backbone oligonucleotide may be contacted with the sample and allowed to hybridise, and then one or more gap oligonucleotides (if used) may be added and allowed to hybridise. Alternatively, all parts of the multi-part probe may be added together.

[0140] The connection includes forming a phosphodiester bond between the 3'OH group at the 3' of the probe and the 5' phosphate group at the 5' end of the probe in two adjacent bases that are hybridized to the target nucleic acid sequence and juxtaposed to connect. Therefore, depending on the design of the probe, a 3'OH group and / or a 5' phosphate group can be provided on a ribonucleotide or a deoxyribonucleotide. Therefore, a ligase that can catalyze the formation of a phosphodiester bond in a target-specific manner can be selected.

[0141] The ligase may be a DNA ligase (i.e., a ligase characterized in that it is capable of catalyzing the formation of a phosphodiester bond between two adjacent deoxyribonucleotides hybridized to a target nucleic acid molecule) or an RNA ligase (i.e., a ligase characterized in that it is capable of catalyzing the formation of a phosphodiester bond between two adjacent ribonucleotides hybridized to a target nucleic acid molecule). As demonstrated in the examples, it has now been shown that both DNA and RNA ligases catalyze the formation of a phosphodiester bond between adjacent 3' ribonucleotides and 5' deoxyribonucleotides hybridized to a target nucleic acid molecule, as well as between adjacent 3' ribonucleotides and 5' ribonucleotides. Therefore, both DNA and RNA ligases may be used in the methods of the present invention. Therefore, reference to a DNA / RNA ligase includes DNA and RNA ligases that are capable of connecting hybridized 3' and 5' nucleotides to form a ligation product comprising a ribonucleotide at or near the site of connection as described herein.

[0142] Exemplary ligases that are particularly useful in the detection methods of the present invention include Chlorella virus DNA ligase (PBCV-1 DNA ligase I), T4 DNA ligase, T4 RNA ligase 1 (T4Rnl1), T4 RNA ligase 2 (T4Rnl2) and DraRN1 ligase. To date, the fidelity of RNA-templated end joining of T4 DNA ligase and PBCV-1 ligase has only been characterized for DNA probes. Surprisingly, when probes as defined herein are used for detection of target nucleic acid sequences, improved efficiency and fidelity of ligation are observed compared to prior art DNA probes alone.

[0143] Suitable ligase and any reagent necessary and / or desired can be combined with reaction mixture, and maintained under the conditions of connection of the oligonucleotide that is sufficient to hybridize.Ligation reaction conditions are well known to those skilled in the art.During connection, reaction mixture can be maintained at about 4 ℃ to about 105 ℃, about 4 to about 80 ℃ in some embodiments, such as about 10 to about 70 ℃, about 15 to about 60 ℃, typically such as about 20 ℃ to about 37 ℃ in the range of temperature and continue at about 5 seconds to about 16 hours, such as about 1 minute to about 1 hour in the range of time.In other embodiments again, reaction mixture can be maintained at about 35 ℃ to about 45 ℃, such as about 37 ℃ to about 42 ℃ in the range, for example, or about 38 ℃, 39 ℃, 40 ℃ or 41 ℃ at a temperature and continue at about 5 seconds to about 16 hours, such as about 1 minute to about 1 hour, including about 2 minutes to about 8 hours in the range of time. In one representative embodiment, the ligation reaction mixture includes 50 mM Tris pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg / ml BSA, 0.25 units / ml RNase inhibitor and 0.125 units / ml T4 DNA ligase. In another representative embodiment, 2.125 mM magnesium ions, 0.2 units / ml RNase inhibitor; 0.125 units / ml DNA ligase are used.

[0144] Obviously, the connection conditions may depend on the ligase used in the method of the present invention. Therefore, the connection conditions described above are only representative examples, and the parameters may be changed according to well-known protocols. However, it should be further understood that changing one parameter (e.g., temperature) may require modifying other conditions to ensure that other steps of the assay are not inhibited or destroyed, such as the binding of the probe to the target nucleic acid molecule. Such operation of the RCA assay method is conventional in the art.

[0145] After forming the ligation probe, it is amplified to increase the copy number of the ligation product in the sample, which can increase the sensitivity of the detection method of the present invention. In some embodiments, a part or part of the ligation probe is amplified. The ligation probe (or part thereof) can be amplified using any convenient method known in the art, such as PCR or its variants, SDA, HAD, LAMP or SMAP.

[0146] In a preferred embodiment, by connecting the circularization probe. Preferably, in such embodiments, the probe can be amplified by rolling circle amplification (RCA). The rolling circle amplification using a circular template to amplify provides a series of RCA products comprising multiple tandem repeats, each of which has a sequence complementary to the circular template oligonucleotide (i.e., a cyclized connection product). RCA can be carried out, for example, by contacting the sample with a separate primer, the primer is complementary to the resulting ring and can hybridize therewith, and the 3' end of the primer can be extended to provide a series of RCA products. Alternatively, the target nucleic acid molecule can include or provide a 3' end of a primer that can serve as an RCA amplification.

[0147] Use DNA polymerase, i.e., the polymerase that can synthesize DNA oligonucleotide from dNTP to carry out the amplification of connecting probe. As used herein, term "DNA polymerase" includes any enzyme that can mix dNTP, and therefore includes enzymes with DNA and RNA polymerase activity. Especially, DNA polymerase can be its main activity or function is to synthesize DNA, but also can mix RNA nucleotide enzyme. Can select to use chimeric DNA-RNA oligonucleotide as the DNA polymerase of amplification template, because connecting probe comprises at least one ribonucleotide. Representative polymerase for step (c) includes Vent DNA polymerase and Bst DNA polymerase.

[0148] Surprisingly, it is also found that Phi29 DNA polymerase can synthesize DNA oligonucleotides using chimeric template oligonucleotides containing ribonucleotides, which includes incorporating deoxyribonucleotides into synthetic products complementary to the ribonucleotides in the target nucleic acid molecule. In other words, Phi29 can act as a reverse transcriptase, and incorporates DNA nucleotides (dNTPs) corresponding to RNA bases complementary to the ribonucleotides in the template. It has been demonstrated that the Phi29 DNA polymerase is used to amplify a connection probe containing up to four consecutive ribonucleotides. In the preferred embodiment of the method of the various methods of the present invention defined above, the DNA polymerase is a Phi29 DNA polymerase. However, this surprising new observed ability of Phi29 to accept RNA-containing substrates has broadened the amplification mediated by Phi29, and in particular the scope of application of RCA, and has opened up new uses for enzymes other than the detection method of the present invention described above. For example, RCA based on Phi29 can be applied not only to nucleic acid detection, but also to oligonucleotide synthesis and library preparation of next generation sequencing technology.

[0149] In one aspect, the invention therefore provides the use of Phi29 DNA polymerase as a reverse transcriptase.

[0150] Alternatively, this aspect also provides a method of synthesizing a DNA molecule, the method comprising contacting a template nucleic acid molecule comprising RNA and DNA bases with a Phi29 DNA polymerase, and generating a DNA reverse complement thereof.

[0151] Although it has been reported that Phi29 DNA polymerase can be converted to have RNA-dependent DNA polymerase activity by introducing specific amino acid substitutions, it has not been demonstrated that wild-type Phi29 DNA polymerase or any other Phi29 sequence variant that has not been modified to increase reverse transcriptase activity may have the ability to function in this manner.

[0152] In particular, the present invention therefore provides the use of Phi29 DNA polymerase as a reverse transcriptase, wherein the sequence of the Phi29 DNA polymerase has not been modified to increase the activity of the reverse transcriptase.

[0153] In addition, the present invention also provides a method for synthesizing a DNA molecule, the method comprising contacting a template nucleic acid molecule comprising RNA and DNA bases with Phi29 DNA polymerase, and generating its DNA reverse complement, wherein the sequence of the Phi29 DNA polymerase has not been modified to increase reverse transcriptase activity.

[0154] In particular, the present invention provides a method for synthesizing a DNA molecule by rolling circle amplification, the method comprising contacting a circular template nucleic acid molecule comprising at least one ribonucleotide and no more than four consecutive ribonucleotides with a Phi29 DNA polymerase, and producing a DNA molecule comprising a plurality of tandem repeats of a reverse complementary sequence of the template nucleic acid molecule, wherein the sequence of the Phi29 DNA polymerase is not modified to increase RT activity.

[0155] Reference to a sequence of a Phi29 DNA polymerase that has not been modified to increase reverse transcriptase activity means that sequence modifications have not been introduced into the Phi29 amino acid sequence for the purpose of introducing (i.e., conferring) or increasing reverse transcriptase activity, which is the activity of an enzyme acting on a template comprising one or more ribonucleotides and incorporating deoxynucleotides complementary to the ribonucleotides in the template into the synthetic product. Thus, the sequence has not been modified to confer or increase the reverse transcriptase activity of the Phi29 DNA polymerase. This does not exclude that the amino acid sequence of the Phi29 enzyme may have other sequence modifications compared to the wild-type or native sequence, but the modifications have not been introduced intentionally or deliberately to confer or increase activity. More specifically, any sequence modifications that may be present relative to the wild-type or native Phi29 enzyme will not confer or increase RT activity.

[0156] By way of representative example, a structure-guided approach for converting Phi29 DNA polymerase to have RNA-dependent DNA polymerase activity is outlined in US 2017 / 0159033, which proposes a number of amino acid substitutions to provide properties such as: increased template polymerase stability or processivity, reduced exonuclease activity, and / or altered template specificity compared to the corresponding parent polymerase. Exemplary amino acids that may be substituted include those that may conflict with RNA / DNA heteroduplexes, or those that may cause steric conflicts between the polymerase and the 2'OH portion of the template, and a number of exemplary substitutions are proposed. Other amino acid substitutions may be those that reduce or eliminate the exonuclease activity of the Phi29 DNA polymerase, substitutions that increase affinity for homologous bases in the active site, substitutions that inhibit binding of the primer strand to the exonuclease domain, substitutions that increase read length and / or processivity, substitutions that reduce interpulse distance, substitutions that enhance thermal stability and / or stability of a binary complex comprising a polymerase and a nucleic acid substrate and / or a ternary complex comprising a polymerase, a nucleic acid substrate, and a homologous nucleotide or nucleotide analog. Thus, preferably, the Phi29 DNA polymerase has not been modified to affect any one or more of the aforementioned properties, and it does not contain any one or more of the exemplary substitutions suggested in US 2017 / 0159033 for converting the Phi29 DNA polymerase into an enzyme having RNA-dependent DNA polymerase activity.

[0157] However, the Phi29 DNA polymerase according to this aspect of the invention may, according to certain embodiments, comprise one or more alternative modifications introduced for different purposes (i.e. in addition to increasing its reverse transcriptase activity), for example in order to alter one or more of its other biochemical properties, and / or to enhance recombinant expression and / or purification of the enzyme (i.e. to increase its yield).

[0158] According to certain embodiments, the sequence of the Phi29 DNA polymerase may not be modified relative to the wild-type sequence. According to a specific embodiment, the sequence of the Phi29 DNA polymerase may therefore be a wild-type or native sequence. In other words, the sequence of the Phi29 DNA polymerase may be a non-mutated sequence.

[0159] In a specific embodiment, the Phi29 DNA polymerase according to this aspect of the invention may have the sequence set forth in SEQ ID NO: 268, or a sequence having at least 95, 96, 97, 98 or 99% sequence identity thereto.

[0160] The template nucleic acid molecule thus comprises at least one ribonucleotide, and the method of this aspect of the invention comprises producing a DNA molecule comprising or having a sequence that is complementary to the template nucleic acid molecule.

[0161] In these aspects, it is preferred that the template nucleic acid molecule comprises no more than 4, and preferably no more than 3 or 2 consecutive ribonucleotides (see above discussion, which also applies to this context). Although better results may be obtained in some cases when the number of consecutive ribonucleotides does not exceed 2, it should be understood that conventional optimization of the method is possible, particularly in the case of circular templates, so that the number of consecutive ribonucleotides does not need to be limited to no more than 2. In some embodiments, the template nucleic acid molecule may therefore comprise 1, 2, 3 or 4 consecutive ribonucleotides.

[0162] This is demonstrated in the examples below.Template molecules can contain ribonucleotides at multiple sites, and the total number of ribonucleotides is not critical or restrictive.Therefore, template nucleic acid molecules can comprise no more than 4 (i.e. 4,3,2 or 1) ribonucleotides at each of two or more separate or different sites.In one embodiment, template nucleic acid molecules can comprise one or more single ribonucleotides, i.e. ribonucleotides flanked by deoxyribonucleotides at its 5' and 3' sides, exceeding a position as described elsewhere herein.Each ribonucleotide site is therefore separated by one or more deoxyribonucleotides therebetween.In some embodiments, two sites can only be separated by a single deoxyribonucleotide.

[0163] In yet another embodiment, the template nucleic acid molecule may comprise ribonucleotides only at a single site within the template nucleic acid molecule. In a specific embodiment, the template nucleic acid molecule may comprise only a single ribonucleotide.

[0164] The consecutive ribonucleotides may preferably be pyrimidine nucleotides rather than purine nucleotides, because it has been demonstrated in the Examples below that Phi29 has better processivity and higher replication accuracy for pyrimidine ribonucleotides (ie, C and U) in the amplification template.

[0165] In this particular aspect of the invention, it is preferred that the template nucleic acid molecule comprises at least one ribonucleotide, rather than a chemical modification, analog or derivative thereof. Thus, in certain preferred embodiments, the ribonucleotide is not a 2'-O-Me modified ribonucleotide, a 2'-F modified ribonucleotide or a locked nucleic acid (LNA).

[0166] Factors necessary for optimal reverse transcriptase activity of the Taq and Bst polymerases described above, such as salts or specific metal cations such as Mn2 + The activity of Phi29 towards longer stretches of ribonucleotides and / or purine RNA bases can be improved by changing the RCA reaction conditions.

[0167] Of course, chimeric template molecules comprising RNA and DNA bases can be produced by connecting probes according to the present invention. However, this aspect of the present invention is not limited to using Phi29 for amplifying connection probes according to the methods herein, and includes any chimeric molecules for amplifying ribonucleotides and deoxyribonucleotides. This chimeric DNA-RNA molecule can be a circular molecule and can be any circular or cyclized probe. Therefore, in any case, Phi29 polymerase can be used to amplify any cyclized probe (e.g., lock probe, iLock, molecular inversion probe, selection probe, etc.) produced in any way. For example, the probe can be a molecular inversion probe that fills a gap with RNA, or a selection probe that hybridizes with a target molecule containing RNA, etc. Therefore, in some preferred embodiments, the template nucleic acid molecule can be a circular template, for example, as formed according to any method disclosed herein.

[0168] Other areas of application may include oligonucleotide production and next generation sequencing as mentioned above, as well as RNA-based therapeutics.

[0169] Generally, any convenient scheme capable of detecting the presence of amplified ligation products can be used in the detection methods of the present invention. The detection scheme may or may not require a separation step. Reference herein to detecting amplified ligation products refers to detecting the sequence formed by the connection of the probe and / or its complement (i.e., its complementary sequence).

[0170] The amplified ligation product (i.e., amplification product) can be detected using any convenient protocol, wherein the particular protocol adopted can detect the amplified ligation product non-specifically or specifically, as described in more detail below. For example, the amplified ligation product can be detected directly, such as using gel electrophoresis, or more preferably by hybridization with a labeled detection oligonucleotide that hybridizes to the amplified ligation product. Alternatively, the amplification product can be detected indirectly, such as by PCR amplification product and the amplification product can be detected.

[0171] Representative non-specific detection schemes of interest include schemes using signal generating systems, which selectively detect single-stranded or double-stranded DNA products, for example, via insertion. Representative detectable molecules that can be used in such embodiments include fluorescent nucleic acid stains (such as phenanthridinium dyes), including monomers or homodimers or heterodimers thereof, which produce enhanced fluorescence when compounded with nucleic acid. Examples of phenanthridinium dyes include ethidium homodimers, ethidium bromide, propidium iodide and other alkyl-substituted phenanthridinium dyes. In another embodiment of the present invention, nucleic acid stains are or incorporate acridine dyes or their homodimers or heterodimers, such as acridine orange, acridine homodimers, ethidium, acridine heterodimers or 9-amino-6-chloro-2-methoxyacridine. In yet another embodiment of the invention, the nucleic acid stain is an indole or imidazole dye, such as Hoechst 33258, Hoechst 33342, Hoechst 34580 (BIOPROBES 34, Molecular Probes, Eugene, OR (May 2000)) DAPI (4',6-diamidino-2-phenylindole) or DIPI (4',6-(diimidazolin-2-yl)-2-phenylindole). Other acceptable nucleic acid stains include, but are not limited to, 7-aminoactinomycin D, hydroxystilbamidine, LDS 751, selected psoralens (furocoumarins), styryl dyes, metal complexes (such as ruthenium complexes), and transition metal complexes (e.g., Tb-incorporated) 3+ and Eu 3+). In certain embodiments of the invention, the nucleic acid stain is a cyanine dye or a homodimer or heterodimer of a cyanine dye that provides enhanced fluorescence when associated with nucleic acids. Any dye described in U.S. Pat. No. 4,883,867 (1989) to Lee, U.S. Pat. No. 5,582,977 (1996) to Yue et al., U.S. Pat. No. 5,321,130 (1994) to Yue et al., and U.S. Pat. No. 5,410,030 (1995) to Yue et al. (all four patents are incorporated by reference) can be used, including nucleic acid stains commercially available from Molecular Probes, Inc., Eugene, Oregon, under the trademarks TOTO, BOBO, POPO, YOYO, TO-PRO, BO-PRO, PO-PRO, and YO-PRO. Any of the dyes described in U.S. Pat. Nos. 5,436,134 (1995) to Haugland et al., 5,658,751 (1997) to Yue et al., and 5,863,753 (1999) to Haugland et al. (all three patents are incorporated by reference) can be used, including nucleic acid stains commercially available from Molecular Probes, Inc. of Eugene, Oregon under the trademarks SYBR Green, EvaGreen, SYTO, SYTOX, PICOGREEN, OLIGREEN, and RIBOGREEN.

[0172] In yet other embodiments of the invention, the nucleic acid stain is a monomeric, homodimeric or heterodimeric cyanine dye incorporating an aza or polyazabenzindolinium heterocycle, such as an azabenzoxazole, azabenzimidazole or azabenzothiazole, which provides fluorescence enhancement when associated with nucleic acids, including nucleic acid stains commercially available from Molecular Probes, Inc., Eugene, Oregon, under the trademarks SYTO, SYTOX, JOJO, JO-PRO, LOLO, LO-PRO.

[0173] In yet other embodiments, a signal generating system that is typically specific to the amplified ligation product rather than the nucleic acid molecule can be used to detect amplification. In these embodiments, the signal generating system can include a nucleic acid or oligonucleotide that specifically binds to a sequence (i.e., a reporter domain sequence) found in the amplified ligation product, wherein the nucleic acid / oligonucleotide can be labeled with a directly or indirectly detectable label. In a specific embodiment, the amplified ligation probe can be detected by ligation sequencing.

[0174] A directly detectable label is one that is directly detectable without the use of other reagents, whereas an indirectly detectable label is one that is detectable through the use of one or more additional reagents, for example where the label is a member of a signal producing system composed of two or more components.

[0175] In many embodiments, the label is a directly detectable label, wherein the directly detectable label of interest includes, but is not limited to, fluorescent labels, radioisotope labels, chemiluminescent labels, etc. In many embodiments, the label is a fluorescent label, wherein the labeling agent used in such embodiments is one or more fluorescently labeled nucleotides, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP), etc. The fluorescent moieties that can be used to label nucleotides to produce labeled probe nucleic acids (i.e., detection probes) include, but are not limited to, fluorescein, cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy630 / 650, etc. As known in the art, other labels, such as those described above, may also be used.

[0176] In certain embodiments, the specifically labeled nucleic acid (detection probe) is labeled with an "energy transfer" label. As used herein, "energy transfer" refers to the process by which the fluorescence emission of a fluorescent group is altered by a fluorescent modifying group. Energy transfer labels are well known in the art, and such labeled oligonucleotide probes include Type probes, as described in U.S. Pat. No. 6,248,526, the disclosure of which is incorporated herein by reference (as well as Held et al., Genome Res. (1996) 6:986-994; Holland et al., Proc. Natl Acad. Sci. USA (1991) 88:7276-7280; and Lee et al., Nuc. Acids Res. (1993) 21:3761-3766). Other examples of detection probes include: Scorpion probes (as described in Whitcombe et al., Nature Biotechnology (1999) 17:804-807; U.S. Pat. No. 6,326,145, the disclosure of which is incorporated herein by reference), Sunrise probes (as described in Nazarenko et al., Nuc. Acids Res. (1997) 25:2516-2521; U.S. Pat. No. 6,117,635, the disclosure of which is incorporated herein by reference), molecular beacons (Tyagi et al., Nature Biotechnology (1996) 14:303-308; U.S. Pat. No. 5,989,823, the disclosure of which is incorporated herein by reference), and conformational assist probes (as described in provisional application serial number 60 / 138,376, the disclosure of which is incorporated herein by reference).

[0177] Thus, any convenient protocol can be used to determine the presence of amplified ligation products. The reaction mixture can be screened, etc. (i.e., assayed, evaluated, assessed, tested, etc.) for the presence of any resulting amplified ligation products to detect the presence of target nucleic acid molecules in the assayed sample. The specific detection protocol can vary depending on the desired sensitivity and application of the method in practice.

[0178] The amplified ligation products can be detected in a variety of different ways. For example, the nucleotides incorporated into the amplified ligation products can be directly labeled, for example, fluorescently or spectrophotometrically in other ways, or radioactively labeled or labeled with any signal generation marker, so that the amplified ligation products are directly labeled. In some embodiments, detection probes as described above, such as fluorescently labeled probes, molecular beacons (as described above), etc. can be used to detect the presence of amplified ligation products, wherein these probes are directed to sequences (e.g., reporter domain sequences) present in the ligation products (i.e., formed by the ligation or formed during the ligation) and therefore only present in their entirety in the amplified ligation products.

[0179] The reaction mixture prepared in this detection step of the method of the present invention may further include an aqueous buffer medium, the aqueous buffer medium comprising a source of monovalent ions, a source of divalent cations and a buffer. Any convenient source of monovalent ions may be used, such as KCl, potassium acetate, ammonium acetate, potassium glutamate, NH4Cl, ammonium sulfate, etc. The divalent cation may be magnesium, manganese, zinc, etc., wherein the cation will typically be magnesium. Any convenient source of magnesium cations may be used, including MgCl2, magnesium acetate, etc. The Mg present in the buffer may be present in the buffer. 2+ The amount may be in the range of 0.5 to 10 mM, but higher or lower amounts may be used and may depend on the type of reaction. For example, for PCR, the amount of Mg2+ present in the buffer may be about 1.5 mM, while for RCA, the amount of Mg2+ present in the buffer may be about 10 mM. Representative buffers or salts that may be present in the buffer include Tris, Tricine, HEPES, MOPS, etc., wherein the amount of the buffer is typically in the range of about 5 to 150 mM, usually about 10 to 100 mM, and more usually about 20 to 50 mM, wherein in certain preferred embodiments, the buffer will be present in an amount sufficient to provide a pH in the range of about 6.0 to 9.5, wherein most preferably pH 7.3 at 72°C. Other reagents that may be present in the buffer medium include chelating agents such as EDTA, EGTA, etc.

[0180] The next step of the method is to detect the signal from the label product of interest, wherein the signal detection may vary according to the specific signal generation system adopted. In some embodiments, only detectable signal (e.g., fluorescence) is determined and used in the subject determination, such as determining or identifying the presence or absence of the connection product of amplification and therefore the target nucleic acid molecule. Depending on the specific label adopted, signal detection can indicate the presence or absence of the target nucleic acid sequence in the target nucleic acid molecule.

[0181] In those embodiments where the signal generating system is a fluorescent signal generating system, signal detection typically includes detecting the change of the fluorescent signal from the reaction mixture to obtain the assay result. In other words, any adjustment of the fluorescent signal generated by the reaction mixture is evaluated. Depending on the nature of the label adopted, the change can be an increase or decrease in fluorescence, but in some embodiments, it is an increase in fluorescence. Any convenient means can be used, such as a suitable fluorometer, such as a thermostable cuvette or a plate reading fluorometer, to screen the fluorescence enhancement of the sample, or, for example, when the sample is a tissue sample on a microscope slide, fluorescence can be detected using a fluorescent microscope. Fluorescence is suitably monitored using a known fluorometer. The signal from these devices (for example, in the form of a photomultiplier tube voltage) is sent to a data processor board and converted into a spectrum associated with each sample tube. Multiple test tubes, such as 96 test tubes, can be evaluated simultaneously. Therefore, in some embodiments, multiple analytes can be detected in parallel, and in other embodiments, multiple analytes can be detected sequentially, such as one analyte at a time or one group of analytes at a time.

[0182] In the case where the detection scheme is a real-time scheme, for example, as employed in a real-time PCR reaction scheme, data can be collected in this manner at frequent intervals throughout the reaction, for example once every 3 minutes. The progress of the amplification reaction can be monitored in various ways by monitoring the fluorescence of the reactive molecules from the sample during each cycle. For example, the data provided by the melting peak can be analyzed, for example by calculating the area under the melting peak, and plotting these data relative to the number of cycles.

[0183] The spectrum produced in this way can be parsed, for example, using the "fitting" of a preselected fluorescent moiety (such as a dye) to form a peak representing each signal generating portion (i.e., a fluorophore). The area under the peak can be determined, the area representing the intensity value of each signal, and if necessary, can be expressed as a quotient of each other. The difference in signal intensity and / or ratio will allow recording the change of the labeled probe in the entire reaction or under different reaction conditions (such as temperature). The change is related to the binding phenomenon between the oligonucleotide probe and the target sequence or the degradation of the oligonucleotide probe in conjunction with the target sequence. The integration of the area under the differential peak will allow calculation of the intensity value of the labeling effect.

[0184] The fluorescence variation of screening mixture provides one or more determination results, and this depends on sample and is to screen once or for example screen repeatedly (for example, as carried out in real-time PCR monitoring) after each circulation of amplified reaction when primer extension reaction ends.The data generated as described above can be explained in various ways.In its simplest form, in the process of amplified reaction or at the end, the amount of the target analyte present in the fluorescence increase or reduction indication sample from sample increases, for example, as associated with the amount of the connection product of the amplification detected in the reaction mixture, shows that amplified reaction has been carried out and therefore actually has the fact of target nucleic acid molecule in the initial sample.Quantitatively also can be carried out by monitoring amplified reaction in the whole amplification process.Quantitatively can also comprise one or more nucleic acid controls in the determination reaction mixture, as described above.

[0185] In this way, the presence of amplification product in (or assessment or determination etc.) reaction mixture can be easily screened, and therefore the presence of one or more target nucleic acid molecules is screened.Described method is applicable to detecting single target nucleic acid molecule and multiple nucleic acid molecule, wherein two or more different target nucleic acid molecules are measured in sample.In these latter multiple situations, the quantity of different probes that can be adopted is typically in the scope of about 2 to about 20 or more, for example, at most 100 or more, 1000 or more etc., wherein a plurality of analytes in sample can be detected in parallel or sequentially.Use a plurality of different probes simultaneously and in single reaction, analyze a plurality of analytes (multiplexing) can be by the sensitivity that increases, and the specificity that increases in some embodiments is strengthened, and described specificity can use method and probe of the present invention to obtain.Each probe group can be designed to produce connection product, and described connection product can be used to determine the presence or absence, quantity and / or position of the analyte that is finally probed.

[0186] The amplified ligated probes can be detected using any well-established method known from the literature for analyzing nucleic acid molecules, including liquid chromatography, electrophoresis, mass spectrometry, microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric analysis (such as ELISA), flow cytometry, mass spectrometry (CyTOF), and the like.

[0187] Detecting the amplified ligated probe further includes determining the sequence of the amplified ligated probe. Therefore, in some embodiments, detection may include sequencing the ligated probe to all or part of the amplification. The probe and method of the present invention can be used homogeneously (i.e. in solution) as described above, or alternatively can be used heterogeneously using a solid phase, for example, wherein the target nucleic acid molecule becomes fixed on a solid phase, thereby allowing the use of a washing step. This may be due to, for example, the fixation of the target nucleic acid molecule in an in situ detection procedure. Using a solid phase assay provides advantages, particularly for the detection of difficult samples: a washing step can help remove unbound and / or unconnected probes, etc., inhibit components, and can enrich target molecules from undesirable large volume samples. Probes of higher concentrations and larger amounts can be used because unbound probes and RNA molecules can be removed by washing.

[0188] In a preferred embodiment of the present invention, the target nucleic acid molecule is detected in situ. This can allow the level, position or location of the target nucleic acid molecule in the direct detection sample. Therefore, the sample can preferably be any sample reflecting the normal or natural ("in situ") location of the target nucleic acid molecule, i.e., any sample of the normal or naturally occurring target nucleic acid molecule. This sample will advantageously be a cell or tissue sample. Particularly preferred are samples such as the following: cultured or harvested or biopsied cell or tissue samples, in which the target nucleic acid molecule can be detected to reveal the location of the target nucleic acid molecule relative to other features of the sample. In addition to cell or tissue preparations, such samples can also include, for example, dehydrated or fixed biological fluids, and nuclear materials, such as chromosome / chromatin preparations, for example, on microscope slides. Samples can be freshly prepared, or they can be pre-treated in any convenient manner, such as by fixing or freezing. Therefore, fresh, freezing or fixed cells or tissues, such as FFPE tissues (formalin fixed paraffin embedded) can be used.

[0189] In alternative embodiments, the target nucleic acid molecule can be fixed. The target nucleic acid molecule is fixed on a solid phase and can be achieved in many ways. Therefore, several embodiments of solid phase assays are envisioned. In one such embodiment, the molecule can first be captured by a fixed (or fixable) capture probe, and the connection product of amplification can be produced so that it is connected to the target nucleic acid molecule, such as by means of a primer for amplification is a target nucleic acid molecule or is connected to the target nucleic acid molecule, as described elsewhere herein. Alternatively, the connection product of amplification can be simply fixed to a solid support. For example, before amplification, the primer for amplification can have a fixable group or part or a fixed member, or can be fixed.

[0190] The fixed capture probe, target nucleic acid molecule, primer for amplification, or the ligation product of amplification can be fixed in any convenient way, i.e., in conjunction with a support. Therefore, it is possible to select a fixation mode and means and a solid support from any number of fixed components and solid supports as widely known in the art and described in the literature according to the selection. Therefore, the capture probe, target nucleic acid molecule, primer for amplification, or the ligation product of amplification can be directly bound to the support (e.g., chemical crosslinking), which can be combined by a linking group, or by one or more intermediate binding groups (e.g., by biotin-streptavidin interaction). Therefore, the capture probe, target nucleic acid molecule, primer for amplification, or the ligation product of amplification can have a component for fixing provided on the support (e.g., affinity binding partner, e.g., biotin or hapten or nucleic acid molecule, which can be combined with its binding partner, i.e., homologous binding partner, e.g., streptavidin or antibody or nucleic acid molecule). The capture probe can be fixed before or after binding analyte. In addition, this "fixable" capture probe can be contacted with a sample and a support.

[0191] Similarly, primers for amplification can be fixed before or after amplification. Capture probes can be, for example, nucleic acid molecules that can specifically bind to target nucleic acid molecules. In other words, capture probes can be fixed (or fixable) probes that are specific to target nucleic acid molecules, and the target nucleic acid molecules include a binding domain that is complementary thereto. Therefore, in this embodiment, first, the target nucleic acid molecules are captured by a fixed or fixable capture probe, the probe is only used for fixing the target nucleic acid molecules on a solid phase, and then the fixed target nucleic acid molecules are subjected to a detection scheme that uses or causes the generation of amplified connection products. More particularly, this capture probe specifically binds to analytes.

[0192] The solid support may be any well-known support or matrix currently in widespread use or proposed for immobilization, separation, etc. These may take the form of particles (e.g. beads which may be magnetic or non-magnetic), sheets, gels, filters, membranes, fibers, capillaries or microtiter strips, tubes, plates or wells, etc.

[0193] The support may be made of glass, silica, latex or a polymeric material. Suitable are materials that present a high surface area for analyte binding. Such supports may have irregular surfaces and may be, for example, porous or granular, such as particles, fibers, fiber meshes, sintered materials or sieves. Granular materials (e.g., beads) are useful due to their greater binding capacity, particularly polymer beads.

[0194] Conveniently, the particulate solid support used according to the invention will comprise spherical beads. The size of the beads is not critical, but they may for example be of the order of at least 1 μm and preferably at least 2 μm in diameter, and have a maximum diameter preferably not exceeding 10 μm, and for example not exceeding 6 μm.

[0195] Monodisperse particles, ie particles of substantially uniform size (eg diameter having a standard deviation of less than 5%), have the advantage of providing very uniform reaction reproducibility. Representative monodisperse polymer particles can be produced by the techniques described in US-A-4336173.

[0196] However, to aid manipulation and separation, magnetic beads are advantageous. As used herein, the term "magnetic" means a support that can impart a magnetic moment (i.e., paramagnetism) when placed in a magnetic field, and is therefore movable under the action of the field. In other words, a support containing magnetic particles can be easily removed by magnetic agglomeration, which provides a fast, simple and effective way to separate the particles after the binding step.

[0197] In another embodiment, the target nucleic acid molecule itself can be fixed (or fixable) on a solid phase, for example by non-specific absorption. In a specific such embodiment, the analyte can be present in cells, optionally fixed and / or permeabilized, and the cells are (capable of) being attached to a solid support, for example a tissue sample containing the analyte can be fixed on a microscope slide.

[0198] As mentioned above, the present invention also provides certain probes for use in the methods of the present invention, namely circularisable probes as defined above, which may be provided in one or more parts. The present invention therefore provides chimeric DNA-RNA padlock probes as defined herein, including invasive padlock probes.

[0199] In one such embodiment, the chimeric DNA-RNA padlock probe is a single circularizable oligonucleotide comprising a first target-specific binding site located at or within the 5' end of the probe and a second target-specific binding site located at the 3' end of the probe, and wherein:

[0200] (i) the second target-specific binding site comprises one or more ribonucleotides;

[0201] (ii) where the first target-specific binding site is internal to the 5' end of the probe, the probe comprises an additional sequence 5' of the first target-specific binding site, such that when the probe hybridizes to the target nucleic acid molecule, the additional sequence forms a 5' flap that does not hybridize to the target nucleic acid molecule and can be removed by cleavage to generate a ligatable 5' end that can be ligated to the 3' end of the probe to circularize the probe, wherein the additional sequence can optionally contain one or more ribonucleotides;

[0202] (iii) The probe, when ligated to form a circle, consists essentially of DNA and comprises no more than 4 consecutive ribonucleotides.

[0203] In another embodiment, the chimeric DNA-RNA padlock probe comprises two or more parts, namely a first part which is a backbone oligonucleotide comprising a first target-specific target binding site located at or within its 5' end and a second target-specific binding site located at its 3' end, and one or more gap oligonucleotides, each of which comprises a target-specific binding site complementary to and capable of hybridizing to the target nucleic acid molecule between the first target-specific target binding site and the second target-specific binding site of the backbone oligonucleotide, and wherein:

[0204] (i) the second target-specific binding site of the backbone oligonucleotide and / or the target-specific binding site of at least one gap oligonucleotide comprises one or more ribonucleotides at their 3′ end;

[0205] (ii) where the first target-specific binding site is internal to the 5' end of the backbone oligonucleotide, the backbone oligonucleotide comprises an additional sequence 5' of the first target-specific binding site such that when the backbone oligonucleotide hybridizes to the target nucleic acid molecule, the additional sequence forms a 5' flap that does not hybridize to the target nucleic acid molecule and can be removed by cleavage to generate a ligatable 5' end that can be ligated to the 3' end of the gap oligonucleotide to circularize the probe, wherein the additional sequence can optionally contain one or more ribonucleotides;

[0206] (iii) one or more gap oligonucleotides optionally comprise an additional sequence 5' of the target-specific binding site, such that when the gap oligonucleotide hybridizes to the target nucleic acid molecule, the additional sequence forms a 5' flap that does not hybridize to the target nucleic acid molecule and can be removed by cleavage to generate a ligatable 5' end that can be ligated to the 3' end of another gap oligonucleotide or the 3' end of the backbone oligonucleotide to circularize the probe, wherein the additional sequence may optionally contain one or more ribonucleotides

[0207] (iv) the backbone and gap oligonucleotides form a loop when ligated, the loop consisting primarily of DNA and comprising no more than 4 consecutive ribonucleotides.

[0208] Such probes may be intrusion lock probes, also referred to herein as iLock probes. In certain embodiments, the probes of the present invention (i.e., probes provided as a single cyclizable oligonucleotide, or portions of probes provided as two or more portions, i.e., backbone oligonucleotides and / or one or more gap oligonucleotides) may therefore comprise an additional sequence 5' of the target binding site of the first target-specific binding site or gap oligonucleotide as described herein, wherein the nucleotides at the 3' end of any additional sequence forming the 5' flap are complementary to the homologous nucleotides identical to the nucleotides at the 3' end (i.e., the ligatable end of the adjacent portion of the probe) of the probe, or backbone oligonucleotides and / or one or more gap oligonucleotides in the target nucleic acid molecule. When such probes hybridize with a target nucleic acid molecule, the nucleotides at the 3' end of the additional sequence forming the 5' flap are therefore prevented from hybridizing with the target nucleic acid molecule by the 3' ligatable end of the probe or backbone oligonucleotides and / or one or more gap oligonucleotides.

[0209] Such invasive probes may be suitable for detecting variant bases in a target nucleic acid molecule. In a probe for detecting variant bases in a target nucleic acid molecule:

[0210] (i) the nucleotide at the 3' end of the probe or backbone oligonucleotide and / or gap oligonucleotide and the nucleotide at the 3' end of the additional sequence described above are complementary to the variant base, and the nucleotide at the 3' end of the additional sequence cannot hybridize with the 3' end of the probe or backbone oligonucleotide or gap oligonucleotide simultaneously with the target nucleic acid molecule, so that the additional sequence can be removed by cleavage to produce a 5' ligatable end of the probe; or

[0211] (ii) the nucleotide at the 3' end of the additional sequence and the nucleotide at the 3' end of the probe or the main chain oligonucleotide and / or the gap oligonucleotide are not complementary to the variant base, so that the nucleotide at the 3' end of the probe, or the main chain oligonucleotide and / or the gap oligonucleotide cannot hybridize with the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3' end of the additional sequence can also be removed by cutting.

[0212] Alternatively, in a probe for detecting a variant base in a target nucleic acid molecule:

[0213] (i) the nucleotide at the 5' end of the first target-specific binding site or the 5' end of the target-specific binding site of the backbone oligonucleotide or the gap oligonucleotide is complementary to the variant base, the nucleotide at the 3' end of the probe or the backbone oligonucleotide and / or the gap oligonucleotide and the nucleotide at the 3' end of the additional sequence described above are complementary to the nucleotide at the 3' position of the variant base, and the nucleotide at the 3' end of the additional sequence cannot hybridize with the 3' end of the probe or the backbone oligonucleotide or the gap oligonucleotide at the same time as the target nucleic acid molecule, so that the additional sequence can be removed by cleavage to produce the 5' ligatable end of the probe; or

[0214] (ii) the nucleotide at the 5' end of the first target-specific binding site or at the 5' end of the target-specific binding site of the backbone oligonucleotide or the gap oligonucleotide is not complementary to the variant base and is also removed by cleavage, thereby creating a gap between the 5' ligatable end and the 3' ligatable end and preventing ligation.

[0215] In a preferred embodiment, the nucleotide at the 3' end of the additional sequence forming the 5' flap is a ribonucleotide.

[0216] In another aspect, the present invention provides a set of invasive probes comprising two or more invasive probes for detecting variant bases in a target nucleic acid molecule, wherein each probe in the probe set comprises a different nucleotide (e.g., A, G, C, or T / U) at a position in an additional sequence and the 3' end of a probe or backbone oligonucleotide or a gap oligonucleotide complementary to a variant base. In certain embodiments, the invasive probe set may comprise three or four probes, each comprising a different nucleotide at the position.

[0217] As discussed above, the total number of ribonucleotides in the probe is not critical, provided that the probe, when ligated, contains no more than 4, or more preferably no more than 3 or 2, consecutive ribonucleotides (see discussion above).

[0218] In certain embodiments, the second binding site at the 3' end of a single moiety probe or at the 3' end of a backbone oligonucleotide, or the target-specific binding site of a gap oligonucleotide may contain no more than 4 or 5 ribonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] The invention may be better understood with reference to the examples and figures, in which:

[0220] Figure 1 The role of RNA nucleotides at the 3' ligatable end at the ligation site using PBCV-1 ligase in the ligation of padlock probes templated by RNA is shown. A: Experimental overview. B: Padlock probes (PLPs) targeting let-7 family members were designed with RNA or DNA terminal 3' nucleotides. The probes were hybridized to matching templates, ligated to PBCV-1 and amplified. The total number of RCA products (RCPs) for each PLP / miRNA pair is shown in the bar graph. The y-axis shows the number of RCPs, while the type of miRNA is depicted on the x-axis. Error bars ± standard error; n=2.

[0221] Figure 2 The effect of the RNA nucleotide at the 3' ligatable end at the ligation site using PBCV-1 or T4Rnl2 ligase is shown. Ligation of 3'-OH(N) / 5'-p(N) relative to 3'-OH(rN) / 5'-p(N) using RNA templates was compared. Intact DNA and chimeric padlock probes were hybridized to the corresponding RNA targets and ligated to (A) PBCV-1 and (B) T4Rnl2. The y-axis shows the number of rolling circle products (RCPs) and the x-axis shows the RNA template used. Error bars ± standard error; n=2. When the probe contained a 3' ribonucleotide at its 3' end, a larger number of ligation products were seen for all target RNAs for PBCV-1, and a substantial increase in the number of ligation products was seen for all target RNAs for T4RnI2 ligase.

[0222] Figure 3 The effect of 3'-OH (rN) mismatch on nick sealing by PBCV-1 and T4Rnl2 ligases is shown. The number of RCPs ( Figure 3 B and 3C) and expressed as a percentage of the iLock probe set for each ligase ( Figure 3 A) indicates.

[0223] Figure 4 The effects of RNA substitutions at various positions in the invasion lock (iLock) probe used in an RNA detection assay using PBCV-1 ligase are shown. Recognition of the invasion structure and structure-specific nucleolytic activity by Taq DNA polymerase can vary with different RNA substitutions. A: Targeting let-7a using the iLock probe, showing the first and second target-specific binding sites of the iLock probe, as well as the arrangement of the 5' additional sequence. RNA nucleotides were introduced at different positions: at the terminal 3' end (3); the 3'-most nucleotide in the 5' flap that competes with the terminal 3' nucleotide at the probe end for target binding (the replacement base, D); the base that becomes the first target binding site of the 5' ligatable end (providing a 5'-phosphorylation donor) after cleavage to remove the additional sequence (iLock probe activation) (5); the entire flap sequence (F). B: Circularization of six iLock designs was evaluated: DNA-only iLock; iLock with the (3) modification (iLock-3); iLock with the (3) and (D) modifications (iLock-3D); iLock with the (3), (D), and (5) modifications (iLock-3D5); iLock with the (3), (D), and (F) modifications (iLock-3DF); and iLock with the (D) and (F) modifications (iLock-DF). The total number of RCPs detected for each iLock probe is shown on the x-axis. Relative to DNA-only iLock, probes containing the (3) modification, and the combination of the (3) modification with the (D) or (D) and (F) modifications showed a substantial increase in the number of RCPs produced. Relative to DNA-only iLock, the combination of the (3) modification with the (D) and (5) modifications, and the (D) and (F) modifications showed a much smaller increase in the relative number of RCPs produced. C: PAGE of iLock DNA, iLock-3 and iLock-3D probes after activation and ligation without (lanes 1-3) and with Taq DNA polymerase (lanes 4-6). The non-activated iLock probe (79) is shortened by 14 nt (65) and ligated after activation (see high molecular weight band at the top of the gel). Lane 4: The band of the activated non-ligated probe is visible (65 nt), the band of the uncleaved probe is clearly visible (79 nt), and only a faint band of the ligated probe is visible. Lane 5: No band of the non-ligated probe is visible, the band of the uncleaved probe is clearly visible (65 nt), and the band of the ligated probe is visible. Lane 6: No band of the non-ligated probe is visible, the band of the uncleaved probe is faint (65 nt), and a strong band of the ligated probe is visible. Together these data show that ribonucleases at the 3' ligatable end of the ligation site improve ligation (lanes 4 and 5) and that ribonucleases at the 3'-most position in the additional sequence cleaved in the invasion assay improve cleavage (lanes 5 and 6).

[0224] Figure 5 Comparison of chimeric and non-chimeric iLock probe ligation is shown. A: Performance of 3D and non-chimeric iLock probes on longer non-miRNA targets. The total number of RCPs for each probe on matching polymorphic templates is shown on the y-axis. B: Comparison of chimeric and non-chimeric iLock probes for miR21 using PBCV-1 and T4Rnl2. The total number of RCPs for chimeric or non-chimeric iLock probes is presented on the y-axis. The ligase used is depicted on the x-axis. Error bars ± standard error; n=2. Both ligases showed improved ligation when chimeric probes were used.

[0225] Figure 6 Ligation efficiency and fidelity of chimeric iLock probes on non-miRNA templates are shown for PBCV-1 and T4Rnl2 ligases. A and B: Fidelity of nick sealing by PBCV-1 and T4Rnl2 ligases on matched polymorphic RNA templates. C and D: Data presented in (A) and (B), but presented as the total number of RCPs generated per iLock probe on each polymorphic template. Error bars ± standard error; n = 2.

[0226] Figure 7 Multiplex detection of let-7 miRNA isoforms using chimeric iLock probes and PBCV-1 ligase is shown. A: miRNA-specific barcodes (NN) are embedded in the probe backbone, between the anchor primer hybridization region and the sequencing library hybridization site. During sequencing, the anchor primer (AP) hybridizes to the RCP, and the sequencing library oligonucleotide pool hybridizes based on the nucleotides at their 5' ends competing with each other. The library containing terminal T is labeled with 3'-FITC; G-3'Cy3 labeling; A-3'Cy5 labeling. Ligase joins the library oligonucleotides corresponding to the barcode base. B: Imaging of the first barcode base by ligation sequencing (SBL).AP: All RCPs stained with AP. Images of each barcode base and merged images are presented. Error bars 5μm. C: miRNAs are mixed in stoichiometric ratios as stated on the x-axis. 1:1:1 represents equal ratios and 0:0:0 represents a no-template control. The total number of reads is shown on the y-axis. Error bars ± standard error; n = number of samples imaged = 2. For each template, the 1:1:3 ratio produced similar numbers of RCPs.

[0227] Figure 8 A comparison of the ligation efficiency of padlock probes and chimeric padlock probes containing 1 (R1pd) or 2 (R2pd) ribonucleotides at the 3' end for PBCV-1 ligase and T4 RN12 ligase using (A) RNA template or (B) DNA template at high and low concentrations is shown. For both ligases, the chimeric padlock probes showed more efficient ligation and amplification than the DNA-only padlock probes for both RNA and DNA targets.

[0228] Fig. 9 In situ detection of KRAS wt and mutant RNA using DNA padlock probes or chimeric padlock probes is shown. A: Microscope images showing detection of mutant and WT RNA using padlock probes (top) or chimeric padlock probes (bottom). B: Average number of mutant RCPs and wild-type RCPs per cell in cell lines A549 and OncoDG1. In both cases, the efficiency of the chimeric padlock probe is much higher. The specificity is high enough to distinguish mutant from wild-type KRAS (more mutant RCPs in A549 and more wild-type RCPs in OncoDG1).

[0229] Fig.10 In situ detection of KRAS wt and mutant RNA using chimeric iLock probes is shown. A: Microscope images showing detection of mutant and WT RNA using chimeric iLock probes. B: Average number of mutant and wild-type RCPs per cell in cell lines A549 and OncoDG1. Specificity is high enough to distinguish mutant from wild-type KRAS (more mutant RCPs in A549 and more wild-type RCPs in OncoDG1).

[0230] Fig.11 Detection of target RNA molecules using gap-filling polymerization and iLock probes is shown. A: The number of RCPs counted in solution after gap-filling polymerization with reverse transcriptase, and Taq cleavage + PBCV-1 ligase ligation (all once) followed by RCA using the chimeric iLock probe. B: In situ detection of RCPs using gap-filling polymerization and the chimeric iLock probe.

[0231] Fig.12 The design of a 2-part iLock probe comprising ribonucleotides at the ends of the backbone and gap oligonucleotides is shown. A target RNA molecule (1) is contacted with a 2-part iLock probe comprising a backbone oligonucleotide (2) and a gap filler oligonucleotide (3). Both the backbone oligonucleotide and the gap filler oligonucleotide comprise an additional sequence (4) at their 5' end that does not hybridize to the target RNA molecule and a ribonucleotide (5) at their 3' end.

[0232] Fig.13 The effect of RNA substitutions on rolling circle amplification using Phi29 DNA polymerase is shown. A: Total amount of RCA generated by the padlock probe (y-axis) in the presence / absence of terminal 3' RNA and in the absence of a synthetic RNA ligation template (template-). B: Rings with 0-7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± standard error; n=2. The same RCA reactions with chimeric rings were also monitored by real-time measurement of Sybr gold incorporation on a qPCR instrument (C and E). C: RCA reaction curves for rings with 0, 1, 2 or 3 RNA substitutions. D: RCPs from C were imaged on a microscope slide, and the size and intensity of individual RCPs were quantified. Black line, median; upper whisker, maximum value within 1.5 interquartile range of the hinge; lower whisker, minimum value within 1.5 interquartile range of the hinge. E: Real-time data for the same RCA reaction as in B, using 0-7 RNA substitutions. Representative samples are presented from replicate experiments. To highlight the initial stages of RCA and to see the difference between samples with low RCA efficiency, fluorescence between 4000 and 6000 is shown.

[0233] Fig.14 Show , chimeric padlock probes containing a 3' ribonucleotide were more easily ligated using PBCV-1 ligase than padlock probes containing a deoxyribonucleotide at their 3' end. Lanes 1-6 - chimeric padlock probes Lanes 7-12 - non-chimeric padlock probes The ligated probe products were expressed as heavier fragments ( * ). For the chimeric probe (lanes 2-3), this is clearly visible after 1-2 minutes, whereas for the non-chimeric probe (lane 12), it is only clearly visible at a later time point.

[0234] Fig.15 A gap-filling padlock probe (2) is shown comprising a ribonucleotide (3) at the 3' end of a 5' additional sequence (4) which is cleaved prior to ligation. The 3' end (5) of the probe hybridized to a target nucleic acid molecule (1) can be extended by gap-filling polymerization.

[0235] Fig.16 A padlock probe (2) is shown that includes a ribonucleotide (3) at a position other than at or near the attachment site (4) that hybridizes to the target nucleic acid molecule (1).

[0236] Fig.17 A padlock probe (2) is shown, comprising a backbone oligonucleotide provided in two parts, and a ligation template (4) which hybridizes to each part of the backbone oligonucleotide for template ligation. As shown, the nucleotide at the 3' ligatable end in this ligation site is a ribonucleotide.

[0237] Fig.18 Amplification rates of circularized padlock probes containing different ribonucleotides are shown. A: Real-time RCA curves of rings containing 1, 2 or 3 continuous RNA substitutions of all four RNA bases are shown. The rate of RCA is monitored by measuring the fluorescence accumulation (y-axis) generated by SybrGold incorporation into RCP. Representative data for each experiment are shown. B: RCA rates of positive controls (pure DNA rings-lower left), negative controls (no rings) and rings with 2, 3, 5 and 7 continuous RNA substitutions, as well as rings of RNA substitutions interspersed between DNA bases are shown. Using rings containing pyrimidine RNA substitutions, Phi29 DNA polymerase exhibits higher RCA rates.

[0238] Fig.19 Shown, in the presence of M-MuLV reverse transcriptase, limited replication of the RNA-rich padlock probe was not recovered. Amplification curves of padlock probes with 0-7 RNA substitutions in the backbone are shown. The rate of RCA is monitored by measuring the fluorescence accumulation (y axis; 3000–30000) generated by SybrGold incorporation into RCP. Replication of rings without additional reverse transcriptase (upper figure) and with additional M-MuLV reverse transcriptase (lower figure) is shown.

[0239] Fig. 20 The stacking diagram of incorporating expected dNTP during RCA reverse transcription is shown. To the padlock probe amplification, monomerization and sequencing containing RNA.RCA monomer is from control DNA ring (upper row), and contains rA, rC, rG and rU at the first RNA position (R1), and contains the ring generation of rUrU, rArA, rCrC and rGrG at its R1 and R2 positions (complete oligonucleotides are in table 9). Align sequencing reads and calculate the frequency of each base in each position.The size of each base is directly proportional to the base frequency.Position R1 and R2 (relative to the RNA position in the padlock probe backbone) are indicated by a frame and highlight position R1 (see arrow).

[0240] Fig.21 In situ detection of ACTB mRNA in cultured human (BjhTERT) and mouse (MEF) fibroblasts is shown. A: Detection of human and mouse ACTB mRNA in BjhTERT and MEF cells using chimeric and non-chimeric chimeric padlock probes (PLPs) and iLock probes. Probes for both targets were included in each sample and show good levels of target specificity. B: The average number of RCPs per cell produced by each probe for each cell line is shown using chimeric and DNA-only PLPs and iLocks. PLP: DNA-only padlock probe; PLPr–3’-(rN)PLP; RiLock: RNA iLock; iLock: DNA-only iLock. For each probe, the signal from the human-specific probe is at the top, while the signal from the mouse-specific probe is at the bottom. In BjhTERT, the human ACTB-specific PLP (top box plot) shows fewer spots than the RNA PLPr (second box plot). Mouse-specific PLP and PLPr do not show signal. For iLock (3rd and 4th box plots), RiLock shows a higher median than iLock (box plots slightly shifted), but the signal is significantly lower compared to PLP. Corresponding data were obtained for MEF mouse cells, where the signal from the mouse-specific probe is higher than that of the human-specific probe.

[0241] Fig. 22 In situ detection of miR21 RNA immobilized on a solid surface is shown. A: miR21 is immobilized and a complementary probe (labeled with a fluorescent dye) is hybridized. The edges of the silicone chamber are intentionally imaged to visualize the immobilization effect; B) When miR21 is not added, the complementary probe does not produce visible fluorescence; mir21 is detected using non-chimeric PLP (C) chimeric PLP (E) iLock probe (D) and chimeric iLock probe (F). The quantitative number of RCPs is expressed as the total number of RCPs / field of view (FOV).

[0242] Fig.23 In situ multiple RNA detection and in situ sequencing in mouse brain tissue sections using chimeric padlock probes are shown. The upper figure shows an overview image of a mouse brain tissue section, where the core is stained in DAPI and the RCA product of the anchor probe staining is produced by chimeric PLPs targeting 18 different neuronal genes (5 probes per gene, each = 90 different probes in total). Below is the area where individual cells are visible in the overview image on the left.

[0243] Fig.24 Phi29 DNA polymerase exhibits higher RCA rates using loops containing pyrimidine RNA substitutions. (A) Real-time RCA curves for loops containing 1, 2, 3, or 4 consecutive RNA substitutions of rG, rU, rA, rC RNA bases are shown (the number of consecutive substitutions is indicated by the curve). The rate of RCA was monitored by measuring the fluorescence accumulation (y-axis) generated by SYBR gold incorporation into RCP. The average fluorescence intensity at each RCA time point was calculated based on repeated experiments. In the presence of Mg 2+ and Mn 2+ RCA was performed in the presence of Mg (solid and dashed lines, respectively). 2+ (solid line) and Mn 2+ (dashed line) shows the linear, early RCA velocity (y-axis) of the PLP from (A). (C) shows the 2+ (solid line) and Mn 2+ (dashed line) RCA of control PLP (non-chimeric DNA circle) in the presence of PLP.

[0244] Fig.25 RCA of chimeric cyclic substrates with RNA substitutions organized in different patterns is shown. The rate of RCA is monitored by measuring the fluorescence accumulation (y-axis) generated by SybrGold incorporation into RCP. 4, 5 and 7 consecutive substitutions (yellow, red, green); 3, 6 RNA substitutions (blue, gray) scattered in 1 or 2 DNA bases and 3 and 8 RNA substitutions (orange, magenta) scattered in a large number of DNA bases (legend only) are introduced into the PLP backbone as shown in the inset (fragments of the backbone fragments only, as indicated by the complete PLP sequence). Average data from repeated experiments. RCA was performed in the presence of magnesium and manganese ions (solid and dashed lines, respectively).

[0245] Fig.26 Show The effect of RNA substitution on the stability of 3'-OH (rG) and 3'-OH (G) padlock probes and ligation with PBCV-1 ligase on RNA is shown. A: PBCV-1 ligase titration. The total number of RCPs generated (y-axis) for each ligase concentration (x-axis) during 30 min ligation. For each time point, the data for the chimeric probes are shown on the left and the data for the non-chimeric probes are shown on the right. B: To assess the stability of the chimeric padlock probes during the first minute of the reaction, a concentration of 26.5 nM (62 mU / / μL) was used. The ligation reaction was terminated by heat inactivating the enzyme at 70°C for 10 min. The total number of RCA products (y-axis) at a given time point (x-axis) is presented.

[0246] Fig. 27 Comparison of chimeric and non-chimeric iLock probe ligation for miR21 and let-7f using PBCV-1 and T4Rnl2 is shown. Total number of RCPs chimeric (left) or non-chimeric (right). iLock probes are presented on the y-axis using PBCV-1 or T4Rnl2 (x-axis). Data for miR21 and let-7f RNA templates are presented.

[0247] Fig.28 The effect of 3'-OH (rN) mispairing on iLock activation and nick sealing fidelity for PBCV-1 and T4Rnl2 ligases is shown. A and B: Performance of chimeric iLock-3D (left) and non-chimeric (right) iLock probes on polymorphic RNA targets. The total number of RCPs for each probe on matching polymorphic templates is shown on the y-axis for A) PBCV-1 DNA ligase and B) T4Rnl2. NC-negative control. C: Fidelity of nick sealing using 3D-type iLock probes on RNA by PBCV-1 DNA ligase (left) and T4Rnl2 (right). The number of RCPs for the same iLock probe on each RNA template is summed and expressed as a percentage within the iLock probe set. The calculated ratio of the expected probe pairs is highlighted.

[0248] Fig.29 Figure 2 shows the ligation efficiency and fidelity of PBCV-1 and T4Rnl2 ligase chimeric iLock probes on polymorphic templates. The total number of RCPs generated and quantified for each iLock probe on each polymorphic template (y-axis) is shown for A) PBCV-1 DNA ligase and B) T4Rnl2. Fig.28 middle.

[0249] Fig.30 dNTP incorporation during RCA reverse transcription is shown. As described in the examples, the padlock probes are monomerized, amplified and sequenced. As depicted in the subsequent separate figures, this method is used to sequence samples with single and double RNA bases. Align the sequencing reads and calculate the frequency of each base in each position. The size of each base is proportional to the base frequency. As indicated in the figure above, the RCA reaction is carried out in the presence of magnesium and manganese ions. The position of the RNA base is indicated by a box. The DNA / RNA sequence originally presented in the PLP sequence is depicted on the right.

[0250] Fig.31 The error incorporation rate at each position in the padlock probe backbone during RCA reverse transcription is shown. The probability of an unexpected nucleotide mismatch at each position for padlock probes with single (left panel) and double RNA substitutions (right panel) is calculated as incorporation error [%] = 1 - expected nucleotide reads / total reads. The average error per base of the sequencing reads (x-axis) and per analyzed sample (y-axis) is shown. RCA reactions were performed in the presence of manganese (A) and magnesium (B).

[0251] Fig.32 The effect of RNA substitutions in circular templates on rolling circle amplification using phi29 DNA polymerase is shown. (A) Rings with 0-7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± standard error; n=2. The same RCA reactions with chimeric rings were also monitored in real time by measuring SYBR gold incorporation on a qPCR instrument (B and C). (B) RCA reaction curves for rings with 0, 1, and 2 RNA substitutions. (C) Real-time data for the RCA reactions using 0-7 RNA substitutions, the same as in B, are shown. Representative samples are presented from repeated experiments. In order to highlight the initial stages of RCA and to show the differences between samples with low RCA efficiency, fluorescence intensity readouts between 3000 and 6000 are presented.

[0252] Fig.33 Rolling circle product analysis based on DNA sequencing is shown, revealing the reverse transcription activity of phi29 DNA polymerase. (A) After RCA, short DNA oligonucleotides are hybridized to the AluI restriction site in the RCA product, and the RCP is digested with the AluI restriction enzyme to obtain RCA monomers. After digestion, the monomers are PCR amplified using primers containing Ilumina adapter sequences. The PCR products are extended using IIlumina index primers. Finally, the sequencing library is prepared using index primer-specific P5 / 7PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated by a green box. (B) Markers showing the sequencing frequency of each position within the RCA monomer generated from a control DNA circle (P1 = dG) and a circle containing a single rG, rU, rA, and rC substitution at the RNA position (P1). Positions P1 and P2 are indicated and position P1 is highlighted with a red box. (C) Incorrect nucleotides are incorporated into each position in the sequenced monomer from (B). The error rates of padlock probes with single (upper panel) and double RNA substitutions (lower panel) are presented, calculated as incorporated error [%] = 1 - number of reads with expected nucleotide / total number of reads. The P1 position of the first RNA substitution is indicated by a box. DETAILED DESCRIPTION

[0253] Example

[0254] Example 1 - Using chimeric DNA / RNA iLock probes, exploiting a novel activity of PBCV-1 DNA ligase: RNA motif Panelized ligation detection of miRNA

[0255] Materials and methods

[0256] Oligonucleotides used in this study

[0257] All oligonucleotides used were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA) using the following synthesis and purification conditions: DNA padlock probes and iLock probes: 4 nM standard desalted DNA oligonucleotides; Chimeric Pad and iLock probes: 4 nM standard desalted RNA oligonucleotides; Decorative probes: HPLC purified DNA oligonucleotides with 5' conjugated fluorophores. All padlock probes were pre-phosphorylated at the 5' end to allow ligation. RNA templates carrying centrally located polymorphic sites are shown in Table 1 (reference oligonucleotides), where polymorphisms are indicated with asterisks.

[0258] The padlock probes are designed so that when the base is paired with the participating RNA target, the terminal arm will form a nicked loop and the discriminating base is located at the 3' end of the probe (Table 1). The miRNA padlock probes used in this study are shown in Table 1. Chimeric padlock probes are ordered with terminal 3'-OH RNA. For comparison purposes, iLock probes were used in this work (Table 2). The standardized chimeric iLock probe design includes a terminal 3' base and a base in the 5' arm that competes with the 3' terminal base for target binding (displacing bases, Figure 4 A, Table 2). Two types of probe barcoding methods were used: traditional and compatible with ligation sequencing readout (iLock probes for chimeric, miRNA-targeted). For traditional rolling circle product (RCP) staining and digital quantification, the reporter sequence was embedded in the sequence connecting the probe arm, separated from the probe arm by a series of 10 adenines (Table 2). For the latter, a common backbone with unique probe-specific barcodes was used (Table 3). To allow barcode decoding, a common anchor primer sequence was embedded in the probe backbone, followed by a sequencing library of two-base barcodes and anchor sequences (Table 3).

[0259] RNA detection assay and digital quantification using amplified iLock and padlock probes

[0260] iLock activation (cleavage) was performed with a 4:1 probe to template excess (typically 2 nM iLock probe was mixed with 0.5 nM RNA template). The duplicate reactions were incubated at 51 °C in a heated lid thermocycler with a volume of 10 μL for 30 min, containing 1 U Taq DNA polymerase (ThermoFisher Scientific), 4 U RNase inhibitor, and 1× Taq polymerase buffer supplied with 8 mM MgCl2. Next, 3 μL of sample volume was transferred to a ligation reaction mixture supplemented with 3.75 U of PBCV-1 DNA ligase (SplintR, M0375S, NEB) or 4 U of T4Rnl2 (M0239S, NEB) in the respective buffers, with a final volume of 15 μL. The reaction was stirred at 37 °C for 30 min. For the padlock probe, the same ligation conditions were applied, but without the activation step. For RCA, 5 μL of the ligation reaction was incubated with 10 nmol of the decorated probe, 0.125 mM dNTP, 0.2 μg / μl BSA, 250 mU Phi29 polymerase (Monserate Biotechnology Group) and 1x 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. Unless otherwise stated, the estimated final concentrations of the amplified products were 5 pM and 20 pM for the padlock probe and iLock probe, respectively. 15 μl of RCA samples were analyzed using an Aquila 400 detection unit (Q-linea, Uppsala). If the RCP concentration was not within the dynamic range of the instrument, the sample was diluted in 4 nM of the decorated probe in 1× labeling 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 re-counted. A template negative reaction was performed in parallel with each experiment as a control.

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

[0262] To test whether chimeric iLock probes can be used to detect changes in miRNA expression in RNA mixtures, we have combined (let-7f):(let-7e):(let-7d) miRNAs in ratios of (1):(1):(1), (3):(1):(1), (1):(3):(1), and (1):(1):(3). The baseline miRNA concentration during the iLock activation step was 0.5 nM, while the baseline miRNA concentration of samples with elevated miRNA concentrations was 1.5 nM. A mixture of 2 nM let-7f, let-7e, let-7d chimeric iLock probes was used, each probe embedded with a unique two-nucleotide barcode (Table 3) and ligation sequencing chemistry sequence. The experiment was performed as described above (using PBCV-1 ligase), except that 10 μL drops of RCA product were spotted on positively charged microscope slides (Superfrost Plus, Menzel ) and evaporated at 55 ° C for 10 minutes. A silicone chamber (Secure-Seal hybridization chamber, Sigma) with a volume of 50 μL was installed above each droplet, and the sample was washed 3 times with 1×TBS. 0.5 μM anchor primer was hybridized in 2×SSC, 20% formamide for 30 min at room temperature. After washing 3 times with 1×TBS, RCP was mixed with a sequencing mixture containing 1×T4 DNA ligase buffer, 10 μg BSA, 1 mM ATP, 0.1 μM sequencing oligonucleotide (Table 3) and 5U of T4 DNA ligase. The slide was incubated at room temperature for 60 min. After washing 3 times with 1×TBS, the silicone chamber was removed, the slide was rinsed with 100% ETOH, air-dried and fixed (SlowFade anti-fading agent, ThermoFisher). The image of RCP was acquired using a 20x objective.

[0263] To visualize the activation and ligation efficiency of various chimeric iLock probes (Table 2), the products were separated by electrophoresis. 5 μM synthetic RNA and 2.5 μM probe were treated as above. After ligation, 50 nM sample was The samples were denatured at 70°C for 3 min, placed on ice for 5 min, and 10 μL was loaded onto a 15% On a TBE-urea gel (EC6885BOX, ThermoFisher Scientific) and in an XCell SureLock TMThe separation was performed for about 90 min at 170 V using a PowerPac basic power supply (Bio-Rad) in a microcell electrophoresis system (ThemoFisher Scientific). The gel was stained for 15 min in 1×TBE running buffer using 1×SybrGold (S11194, Invitrogen) and then imaged in a Gel Doc XR System (Bio-Rad). In the chimeric probe binding assay, a certain concentration of template and padlock probe as stated above was incubated at room temperature for 10 minutes with 62 mU / μL PBCV-1 DNA ligase and 0.4 U / μL RNase inhibitor. The reaction was terminated by adding 1 μL 0.5 M EDTA and 5 μL 100% formamide.

[0264] result

[0265] Effects of 3'-OH RNA on RNA-dependent ligation by PBCV-1 DNA ligase: RNA end ligation on different RNA substrates

[0266] We compared the ability of PBCV-1 ligase to circularize padlock probes hybridized to let-7a, where the 5' end of the probe is DNA and the 3' end is either DNA or RNA. While the ability of T4Rnl2 to ligate a chimeric 3' RNA acceptor strand to a 5' donor strand on RNA has been well characterized, this has only been demonstrated for PBCV-1 DNA ligase on a DNA template. By PAGE separation, we compared the ligation efficiency of the chimeric padlock probe relative to the DNA padlock probe over reaction time ( Fig.14 In addition, we measured ligation efficiency as the total number of rolling circle amplification products (RCPs), digitally counting each padlock / template pair ( Figure 1 A).

[0267] When PLP containing 3' RNA is ligated to the miRNA target, PBCV-1 ligase catalyzes efficient end joining ( Figure 1 B). For longer non-miRNA targets, chimeric and non-chimeric probes have similar ligation efficiencies ( Figure 2 A). Because the presence of RNA results in greater nucleic acid duplex stability, we hypothesized that more stable duplexes would be ligated more quickly during the initial reaction phase. T4Rnl2 efficiently ligated chimeric padlock probes, whereas DNA padlock probes were relatively inactive ( Figure 2 B). Our finding that PBCV-1 readily accepts chimeric padlock probes as substrates prompted us to systematically characterize ligation fidelity on synthetic targets with polymorphic positions in the centrally located nucleotide ( Figure 3To measure the effect of mismatched chimeric substrates on the end-joining activity of PBCV-1 and T4Rnl2 ligases, chimeric padlock probes differing in their terminal 3' nucleotides (rA, rU, rG, rC) were each hybridized to four different RNA targets, ligated, and amplified by RCA ( Figure 3 PBCV-1 ligase is highly tolerant to most 3' RNA mismatches ( Figure 3 On the other hand, T4Rnl2 had moderate accuracy in joining rC / rG (82%) and rA / rC (64%), but had poor fidelity in joining the ends of other combinations ( Figure 3 A, 3C).

[0268] Effects of various RNA substitutions on RNA-templated iLock probe activation, PBCV-1 and T4Rnl2 ligation efficiency and fidelity

[0269] In our previous studies, we exploited the structure-specific 5' flap cleavage activity of Taq DNA polymerase for invasion assays to activate padlock probe molecules for ligation. We have shown that this iLock probe assay increases the fidelity of ligase-based RNA detection (Krzywkowski, supra). Because the PBCV-1 ligase is fairly tolerant to most chimeric 3' mismatches tested, we tested how the presence of RNA substitutions in different positions of the iLock probe would affect probe activation and ligation compared to the DNA iLock probe. Multiple iLock probes targeting let-7a miRNA were designed (Table 2) containing RNA substitutions in various probe positions ( Figure 4 A). One chimeric probe (referred to as "3") has an RNA substitution at the 3' end. In the "3D" probe, the replacement bases of the terminal 3' and 5' flaps are replaced by RNA. The "3D5" probe has an additional RNA base at position 3' of the "D" position in addition to the substitution in the "3D" probe. Upon successful activation of iLock, this RNA base will become the 5'-phosphate donor end in the ligation reaction. Finally, we designed probes with the terminal 3' and the complete 5' flap as RNA bases ("3DF"), as well as probes in which only the 5' flap is composed of RNA ("DF") (i.e., lacking 3' RNA).

[0270] Compared with the non-chimeric let-7a iLock, iLock-3 significantly increased the detection of let-7a miRNA. Figure 4 C), under given conditions, only a small fraction of non-chimeric iLock probes are activated (cleaved), and an even smaller fraction is ligated ( Figure 4C, lane 4). Almost all activated iLock-3 probes have been ligated, e.g. Figure 4 Quantitative gel shift in C (lane 5) and the total number of RCA products generated by the iLock-3 probe ( Figure 4 When the flap nucleotide displaced by the invading 3' end RNA was replaced by RNA, as in iLock-3D, additional efficiency gains were observed. Most iLock-3D was activated and ligated ( Figure 4 C) A similar effect was observed for the iLock-3DF probe, where the entire 5' flap sequence was RNA, whereas the positive effect was lost in the absence of the terminal 3' RNA base ( Figure 4 B). Interestingly, the iLock-3D5 probe containing 3'-(rN) / 5'-(rN) after activation showed significantly lower performance than the iLock probe with a deoxyribonucleotide at the (5) position. For other miRNAs (miR21) tested using PBCV-1 and T4Rnl2 ligase, a significant improvement in the performance of the iLock-3D5 probe was observed ( Figure 5 ). Similarly, in repeated experiments using other iLock-3D probes, improved performance was observed for PBCV-1 and T4Rnl2 using let-7f as a template ( Fig. 27 To test whether the accuracy of RNA sensing is maintained using chimeric iLock-3D probes, we used four chimeric iLock-3D probes to target four polymorphic RNA templates (Table 2). The chimeric iLock probes showed excellent fidelity for the matched rC / rG, rA / rU, and rU / rA probe pairs ( Figure 6 ). When the template was omitted, the iLock-3D probe showed no ligation product. T4Rnl2 showed full compatibility with the iLockRNA detection assay, easily ligating the target-matched 3'-OH(rN) / 5'-p(N) iLock probe ( Figure 6 B). For PBCV-1 and T4RN12, the rG / rC pairs were detected with relatively low fidelity, showing rG / rU mis-ligation at 5% and 27%, respectively ( Figure 6 In another experiment, both PBCV-1 and T4Rnl2 showed full compatibility with the iLockRNA detection assay, allowing easy ligation of the target-matched 3'-OH(rN) / 5'-p(n) iLock probe ( Fig.28 AB, Fig.29 , AB). For both enzymes, rG / rC pairs were detected with relatively low fidelity, showing that rG / rU mismatches were 21% and 11%, respectively. Fig.28AB It can be seen that the chimeric ilock has better performance than the DNA ilock.

[0271] Multiplexed detection of let-7 isoforms using chimeric iLock probes

[0272] High multiplexing capability is one of the most advantageous functions of padlock probes. The distinction of amplification products derived from different padlock probes is typically achieved by using unique probe-specific decorative oligonucleotides (labeled with fluorophores with different emission spectra). Alternatively, a unique barcode sequence can be embedded in the padlock probe backbone, which can be decoded using a new generation of connection sequencing chemistry. In order to evaluate the compatibility of chimeric iLock probes with connection sequencing readout, we redesigned four let-7 family iLock-3D probes, as described in the method section (Table 3). In order to evaluate whether the barcoded iLock-3D probe can be used for multiple miRNA analysis, we combined let-7f, let-7e, let-7d miRNA with four different stoichiometric ratios. Ideally, the ratio will be accurately reflected in the miRNA specific sequencing read. The iLock probe is multiplexed and the amplified product is fixed on a glass surface. The barcode of RCP is decoded using a connection sequencing chemistry. Because only three iLock probes were used in this experiment, it is sufficient to sequence the first barcode position to decode the detected miRNA. iLock probes showed similar relative efficiencies for miRNA pools ( Figure 7 In samples with increasing concentrations of one miRNA, the signal of the corresponding iLock probe increased, while the signals of other targets remained stable ( Figure 7 C).

[0273] Example 2 - Ligation of a DNA padlock probe and a chimeric probe comprising 1 or 2 ribonucleotides at its 3' end.

[0274] Materials and methods

[0275] The ligation reaction was performed with a DNA padlock probe or a chimeric padlock probe with 1 or 2 terminal 3' ribonucleotide bases at a final concentration of 1 nM, and a synthetic KRAS RNA template or KRAS DNA template at a final concentration of 2 nM. The oligonucleotide sequences are shown in Table 6. The reaction was incubated at 37°C for 30 min in a final volume of 10 μL in a ligation buffer containing 1 U / μL RNase inhibitor, 0.2 mg / mL BSA and 1x SplintR buffer or T4 RNA ligase II buffer and 0.25 U / μL (low concentration) or 1.25 U / μL (high concentration) SplintR ligase, or 0.2 U / μL (low concentration) or 1 U / μL (high concentration) T4 RNA ligase II. Afterwards, the ring was amplified by rolling circle amplification in RCA reaction buffer as described above, with a final ring concentration of 100 pM. Finally, the RCA product was labeled with a Cy3-labeled detection probe at a final concentration of 10 pM. Labelled RCA products were counted digitally.

[0276] result

[0277] For both SplintR ligase and T4 RNA ligase II, and on both RNA and DNA templates, both chimeric padlock probes with 1 or 2 terminal 3' ribonucleotide bases produced more RCA products than pure DNA padlock probes ( Figure 8 ). Increasing ligase concentration did not have any effect on chimeric padlock probes, but had a slight negative effect on DNA padlock probes. There was no difference in RCA product counts between chimeric padlock probes with 1 or 2 terminal 3' ribonucleotide bases.

[0278] On RNA templates, Splint R ligase and T4 RNA ligase II have similar activities ( Figure 8 A). On DNA templates, the differences between DNA padlock probes and chimeric padlock probes for SplintR ligase were similar to those on RNA templates, but a very robust increase in RCP counts was recorded for ligation of chimeric padlock probes on DNA templates using T4 RNA ligase II ( Figure 8 B) T4 RNA ligase II does not accept 3 when templated by DNA ' DNA and 5 ' DNA termini, but readily accept 3 ' Probes at the ends of RNA.

[0279] In summary, the use of chimeric probes makes ligation reactions using SplintR ligase and T4 RNA ligase II on RNA templates more efficient than conventional DNA probes. Chimeric probes allow T4 RNA ligase II to be used for ligation reactions on DNA templates.

[0280] Example 3 - In situ KRAS point mutation detection using chimeric padlock probes and chimeric iLock probes

[0281] Materials and methods

[0282] ONCO-DG-1 and A-427 cell lines were cultured in RPMI medium without L-glutamine supplemented with 10% FBS 2mM L-glutamine and 1x penicillin-streptomycin (PEST). A-549 was cultured in DMEM supplemented with 10% FBS and 1xPEST. When confluent, all cell lines were inoculated on Superfrost Plus slides and attached for 12h. The cells were then fixed at room temperature for 15min in 3% paraformaldehyde in DEPC-treated PBS (DEPC-PBS). After fixation, the slides were washed twice in DEPC-PBS and dehydrated for 4min by 70%, 85% and 100% ethanol series, respectively. The safety sealed chamber was mounted on a slide, and the cells were hydrated by simple washing with PBS-T (DEPC-PBS with 0.05% Tween20), and then permeabilized for 1min at room temperature with 0.1HCl in H2O. Cells were washed twice in DEPC-PBS-T, and then DNA or chimeric probes were added to hybridization buffer containing 2x SSC, 20% formamide and 0.4U / μL RNase inhibitor at a final concentration of 50nM. Oligonucleotide sequences are shown in Table 6. Probes were hybridized for 60min at 37°C. The probe hybridization mixture was then removed and cells were washed for 15min at 37°C in preheated (37°C) wash buffer containing 2x SSC and 25% formamide, and washed once at 37°C in preheated (37°C) wash buffer containing 2x SSC and 20% formamide for 15 minutes. Cells were washed once in PBS-T. The ligation reaction mixture was then added to the DNA / chimeric padlock probe experiment ( Fig. 9 ) and adding the invasion reaction mixture to the DNA / chimeric iLock assay ( Fig.10). The ligation reaction mixture contained SplintR ligase buffer, 0.2mg / mL BSA, 0.8U / μL RNase inhibitor, and 0.25U / μL SplintR ligase. The ligation reaction was incubated at 37°C for 60min. The invasion reaction mixture contained the same as the ligation reaction and contained an additional 0.1U / μL Taq DNA polymerase. The invasion reaction mixture was incubated at 37°C for 60min. Subsequently, all experimental reactions were washed twice with PBS-T. 100nM RCA primers were hybridized in situ in 2x SSC and 20% formamide hybridization buffer at room temperature for 30min. The cells were washed twice in PBS-T. Next, the RCA reaction mixture containing 1x phi29 reaction buffer, 0.25mM dNTPs, 0.2mg / mL BSA, 1U / μL phi29 polymerase, and 5% glycerol was added and incubated at 37°C for 3 hours. Subsequently, the cells were washed twice in PBS-T, and the detection probes were hybridized in situ with the RCA products (Cy3-labeled probes with KRAS wild-type probes, Cy5-labeled probes with KRAS mutant probes) in 2x SSC and 20% formamide hybridization buffer for 30 min at room temperature. The cells were washed three times in PBS-T, the nuclei were stained with DAPI, washed three times again, and then fixed in Slowfade fixation medium. The cells were imaged under a fluorescence microscope with a 20x objective and the RCA products were quantified using Cellprofiler software.

[0283] result

[0284] Compared with DNA padlock probes, chimeric padlock probes have higher in situ RNA detection efficiency for both A549 and OncoDG1 cell lines ( Fig. 9 B). In addition, the chimeric padlock probe increased the ratio of specific / nonspecific RCPs per cell in both A549 and OncoDG1 compared to the DNA padlock probe (more mutant RCPs were detected than wild-type RCPs in A549 cells (carrying KRAS codon 12 point mutations), and more wild-type RCPs were detected than mutant RCPs in OncoDG1 cells (KRAS wild-type)), making it possible to more accurately detect point mutations in situ directly on RNA using the chimeric probe.

[0285] To further improve the specificity of point mutations, we applied the chimeric iLock probe in situ and found that KRAS wild-type mRNA was specifically detected in OncoDG1 cells and KRAS codon 12 point mutation was detected in A549 cells ( Fig.10 ).

[0286] In summary, chimeric probes significantly improve in situ RNA detection efficiency, making in situ RNA analysis more sensitive, cost- and time-efficient than classical cDNA methods. In addition, chimeric padlock probes, and especially chimeric iLock probes, show higher specificity compared to DNA padlock probes, making in situ RNA analysis more efficient and accurate.

[0287] Example 4 - Gap Filling iLock Probe

[0288] Materials and methods

[0289] iLock reaction for gap filling in solution:

[0290] The ligation reaction was performed with a final concentration of 10 nM gap-filled ILock probe and a final concentration of 30 nM synthetic KRAS RNA template (or no template in the negative control). The oligonucleotide sequences are shown in Table 7. The reaction was continued at 37 ° C for 60 min in a final volume of 10 μL in a reaction buffer containing 1 U / μL RNase inhibitor, 1 U / μL reverse transcriptase, 25 μM dNTP (or no dNTP in the gap-filled negative control), 0.2 mg / mL BSA, 1xSplintR ligase buffer, 0.1 U / μL Taq DNA polymerase and 0.25 U / μL SplintR ligase. Afterwards, as described above, the ring was diluted 10 times in PBS-T (to a theoretical concentration of 1 nM) and then amplified with rolling circle amplification in RCA reaction buffer, with a final ring concentration of 100 pM. Finally, the RCA product was labeled with a Cy3-labeled detection probe at a final concentration of 10 pM and digitally counted.

[0291] In situ gap filling iLock reaction

[0292] OncoDG1 cells were prepared and treated as described in Example 3. The gap-filling iLock probe was hybridized in situ with KRAS RNA at a concentration of 50 nM under the same conditions as in Example 3. After washing, the gap-filling polymerase invasion mixture was added to the cells containing 1 U / μL RNase inhibitor, 10 U / μL reverse transcriptase, 25 μM dNTPs (or no dNTPs in the gap-filling negative control), 0.2 mg / mL BSA, 1x SplintR ligase buffer, 0.1 U / μL Taq DNA polymerase, and 0.25 U / μL SplintR ligase and incubated on the cells at 37°C for 60 min. The cells were washed twice in PBS-T. Next, an RCA reaction mixture containing 1x phi29 reaction buffer, 0.25 mM dNTPs, 0.2 mg / mL BSA, 1 U / μL phi29 polymerase, and 5% glycerol was added and incubated at 37°C for 3 hours. Subsequently, cells were washed twice in PBS-T and Cy3-labeled detection probes were hybridized to RCA products as described above. Cells were washed three times in PBS-T, nuclei were stained with DAPI, washed three times again, and then fixed in Slowfade mounting medium. Cells and RCA products were imaged under a fluorescence microscope with a 20x objective and RCA products were quantified using Cell profiler software.

[0293] result

[0294] The RCA products of the in-solution gap-filling polymerization-invasion reaction were quantified and plotted on Fig.11 In A. When RNA template was present, RCA products were counted, indicating that the reaction proceeded adequately. When template was absent (template negative), significantly lower numbers of RCPs were counted, indicating that the gap-filling iLock probe could not be extended and therefore could not be removed. ' Flap and cannot connect to 3 ' The end.

[0295] In control reactions in which RNA template was present but no dNTPs were added, significantly lower numbers of RCPs were counted compared to positive reactions (with template and dNTPs), indicating that gap filling by polymerization and triplex formation was limited, significantly reducing cleavage of the flap and thus generating fewer ligation products ( Fig.11 A). The same trend is seen in the in situ reaction ( Fig.11 B and C).

[0296] Example 5 - Use of Phi29 as a reverse transcriptase

[0297] Materials and methods

[0298] Oligonucleotides

[0299] The oligonucleotide sequences are shown in Tables 8 and 9. The probes are provided as described in Example 1. The ligation reaction is performed on a synthetic KRAS mRNA template. The padlock probe is designed so that after RNA hybridization, the probe is circularized, thereby forming a nick between the terminal arms. In order to conveniently assess the size of the rolling circle product (RCP), a reporter sequence is embedded in the sequence connecting the probe arm (main chain). By hybridizing with the reporter sequence, a complementary decoration is used for RCP staining. For real-time RCA assessment, the amplified DNA is stained with SybrGold dye.

[0300] Real-time RCA. In order to evaluate the effect of RNA base on the reverse transcription performance of Phi29 polymerase, 20nM padlock probe was mixed with 10nM RNA template supplemented with 4U RNase inhibitor (DNA Gdansk), 3.75U PBCV-1 DNA ligase (SplintR, M0375S, NEB) in a final volume of 15μL. The reaction was stirred for 30min at 37°C. After connection, 2μL connection volume (ring) was mixed to a final concentration of 2nM ring in 18μL RCA reaction mixture containing 1x Phi29 reaction buffer (Thermo Fisher), 125μM dNTP (DNA Gdansk), 0.2mg / mL BSA (NEB) and 1×SybrGold (S11194, Invitrogen). In order to ensure that RCA in all samples is triggered at the same time, the ring is placed in a test tube cap and spun into a pre-processed master mix using a desktop centrifuge. RCA was initiated immediately and SybrGold incorporation was detected using the Mx3005P qPCR system (Agilent Genomics) at 37°C for 60 min, followed by Phi29 inactivation at 65°C for 2 min.

[0301] To investigate whether the RCA efficiency of RNA-rich circles could be stimulated by the addition of reverse transcriptase, 100 U of RNaseH(-)TranscriptME reverse transcriptase (DNA Gdansk) was added to the RCA reaction mixture.

[0302] Sequencing of RCA products generated from RNA-containing circles

[0303] Monomerization

[0304] In order to sequence the bases incorporated into the RCA products corresponding to the RNA bases in the circular template, the RCA products are first monomerized by restriction digestion. First, the RCA products measured from real-time RCA as described above are diluted to a concentration of 100pM in PBS-Tween 0.05%. Then, in a reaction mixture containing 1x Phi29 DNA polymerase buffer, 2mg / mL BSA, 100nM restriction oligonucleotides (AluI KRAS RO-Table 9), 120mU / μL AluI (NEB) and 10pM final concentration of RCA products, the RCA products are digested with AluI restriction endonucleases during 10min incubation at 37°C and then heat-inactivated at 65°C for 2 minutes. After completely digesting 10pM RCA products, the RCA monomer concentration is about 10nM (1 hour RCA of 80 base rings produces about 1000 times of amplification). RCA monomers were diluted to 100 pM in PBS-Tween 0.05%.

[0305] Sequencing of library preparations of RCA monomers

[0306] During the PCR reaction, the RCA monomer was first labeled with the Illumina adapter sequence, and the PCR reaction contained 1x TaqDNA polymerase buffer (NEB), 1.5mM MgCl2 (NEB), 250μM dNTP, 1x SybrGold, 25mU / μL Taq DNA polymerase (NEB), 0.5μM forward primer PE1 (Table 9), 0.5μM reverse primer PE2 (Table 9) and a final concentration of 10pM RCA monomer. The PCR reaction started with a 5min denaturation at 95°C and cycled 20 times between 95°C for 15 seconds, 55°C for 30 seconds and 70°C for 20 seconds. The reaction was monitored in a qPCR instrument and stopped before the amplification reached saturation. After the first PCR step (extension step), 1 μl of PCR product was spiked into a culture medium containing 1x 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 labeled with a unique combination of one of 7 different forward primers and one of 3 different reverse primers), and programmed at 95°C for an initial 2 min, and 2 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 1 min, and an additional cycle of 72°C for 3 min. The indexed PCR products were diluted 200-fold into a PCR mix containing 1x Phusion HF buffer (ThermoScientific), 0.2 mM d(A,T,G,C)TP (Thermo Scientific), 1% DMSO, 500 nM P5 and P7 primers, and programmed at 95°C for 2 min, and 15 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. PCR products were pooled and purified using a QIAquick PCR purification kit and 550 system (Illumina) 500hi-Output KT v2 (75CYS) sequencing. Reads containing the correct primer sequence at the expected position were extracted and analyzed using WebLogo.

[0307] Morphological assessment of RCP size and intensity

[0308] In order to measure the size and intensity of RCA products, the RCA products from the real-time RCA reaction were diluted to a final concentration of 20pM and labeled with a final decorative probe concentration of 5nM under standard hybridization conditions. 10 μL of fluorescently labeled RCA products were applied to Superfrost glass slides (Thermo Fisher), spread by 20x20mm coverslips (Menzel) and electrostatically bound to the positively charged surface during 15 minutes of incubation. The coverslips were removed, the slides were briefly washed in PBS, fixed in a fixing medium, and imaged with 20 times magnification in the Cy3 channel on a Zeiss Axioplan fluorescence microscope. The images were exported as original black and white (BW) pictures and processed using Cell Profiler software. In brief, each image was pre-processed using automatic high hat filtering. Objects were identified using manually adjusted thresholds and separated based on object intensity. Average fluorescence intensity and object size were recorded, exported as csv files and processed in R! Studio.

[0309] result

[0310] Phi29 DNA polymerase accepts chimeric circles as rolling circle amplification (RCA) templates

[0311] We have observed that circular chimeric padlock probes containing both RNA and DNA nucleotides can be used as substrates for RCA, indicating that Phi29 DNA polymerase has reverse transcriptase activity. To investigate this activity, we have circularized various RNA / DNA chimeric padlock probes containing 1-7 RNA substitutions in the DNA probe backbone and used the circularized probes as templates during RCA ( Fig.13 ).

[0312] The RCA reaction was monitored in real time by SybrGold incorporation. Additionally, the RCA products were digitally counted and assessed for size and intensity (ie morphology).

[0313] We observed that PBCV-1 readily encapsulated pure DNA probes and chimeric 3 ' -(rN) / 5 ' (N) The probe is nicked, and Phi29 polymerase accepts the pure DNA and RNA-containing circle as a template for RCA ( Fig.13 A) From 3 ' The higher number of RCA products for the RNA probes is likely due to the increased ligation efficiency of the chimeric probes, as detailed in Example 1. When no template is added during the ligation reaction, the probes are not ligated and cannot be amplified ( Fig.13 A, target-).

[0314] Next, we aimed to investigate the RCA efficiency of chimeric DNA / RNA circles in the absence of ligation reaction bias. To this end, we added RNA substitutions to the ring backbone that do not participate in the ligation reaction. Thus, all probes contained the same target-complementary DNA probe arm sequence (which contributes to the formation of ligation substrate) and were ligated to the same RNA target. We then calculated the efficiency of RCA by counting the RCA products ( Fig.13 B) and monitor the amplification reaction in real time ( Fig.13 C) to study how increasing numbers of RNA substitutions affect the RCA reaction efficiency. Padlock probes with sequences SEQ ID NO: 119, 120, 124, 131, 110, 111 and 112 and chimeric probes with 0, 1, 2, 3, 4, 5 and 7 consecutive ribonucleotides were used as DNA probes.

[0315] We observed no effect on RCA efficiency when replacing a single RNA in the probe (loop) backbone ( Fig.13 B and 13C, Fig.32 A and 32B). When the loop was replaced by 2 consecutive RNA nucleotides, a strong inhibition of RCA (approximately 90%) was observed ( Fig.13 B and 13C, Fig.32 A and 32B). For loops with more than 2 RNA substitutions, no amplification was detected ( Fig.13 E, Fig.32 C). When rolling circle products (RCPs) were imaged using epifluorescence microscopy, the average size and intensity of RCPs for rings with 2 consecutive RNA substitutions decreased ( Fig.13 D). More than two consecutive RNA substitutions (3-7) resulted in complete inhibition of RCA and no RCP was detected in these samples.

[0316] We additionally investigated whether RCA of RNA-rich circles could be restored by supplying M-MuLV reverse transcriptase during the RCA reaction. However, under the reaction conditions used, RCA activity was not restored in the presence of reverse transcriptase. In contrast, the amplification rate was significantly reduced by adding M-MuLV reverse transcriptase (lower graph of the curve) compared to the RCA reaction without additional reverse transcriptase ( Fig.19 ).

[0317] Phi 29 DNA polymerase preferentially reverse transcribes RNA pyrimidines during RCA

[0318] In previous experiments, DNA bases in the loop backbone were increasingly replaced by RNA bases. Strong inhibition of amplification was observed for loops with rGrA and rGrArC substitutions. To investigate whether there is sequence dependence in the RCA efficiency of RNA-containing loops, we monitored RCA rates in real time using loops with single rU / rA / rC / rG RNA bases as well as di- and tri-nucleotide long homologous nucleotide stretches (Table 9). We observed efficient RCA for all single RNA substitutions ( Fig.18 A). For dinucleotide RNA substitutions, we observed the highest RCA rate for the rCrC loop, followed by the rUrU loop, while the rArA and rGrG loops were significantly suppressed. For trinucleotide RNA loops, only the rCrCrC loop produced detectable RCA, but its rate was significantly slower than that of the loop containing rCrC ( Fig.18 A). Many ribonucleotide probes SEQ ID NOs: 108-112, 124-127, 131-134, 137 and 139 contain additional isonucleotide ribonucleotide sequences. The experiment was repeated using probes SEQ ID NOs: 267-281 (Table 13) without the additional ribonucleotide stretches and similar results were observed ( Figure 24-25 ).

[0319] To investigate whether RCA could be restored for longer mixed RNA / DNA segments, we inserted 1 and 2 DNA bases into segments of 3 and 6 RNA bases, respectively ( Fig.18 B). Circles containing the rGArCGrU sequence in the backbone were amplified, whereas no RCA was detected for circles with 6 inserted RNA substitutions, or for circles with 5 and 7 consecutive RNA bases ( Fig.18 B).

[0320] Manganese ions increase the RNA-dependent RCA activity of phi29 DNA polymerase

[0321] Because some DNA-dependent DNA polymerases 2+ In the case of reverse transcription RNA, we 2+ and Mn 2+ The phi29 DNA polymerase RCA rates were compared in the presence of Mn 2+ As cofactors, in addition to rC, phi29 DNA polymerase also efficiently amplifies mono-, di-, and tri-nucleotide stretches of rU and rA ( Fig.24 Interestingly, the expansion rates of rCrC, rUrU, and rArA loops were higher than those of loops with single RNA substitutions, and for Mg 2+ The same is true for rCrC as a cofactor ( Fig.24A, B). To investigate whether RCA can be restored if multiple RNA bases are mixed with DNA bases, Mg 2+ and Mn 2+ Multiple chimeric constructs were amplified ( Fig.25 ). Based on our observations, phi29 DNA polymerase is able to engage in efficient Mn 2+ Dependency RCA ( Fig.25 , Table 12). Interestingly, circular chimeric substrates with up to 8 RNA bases were fully amplified when the substitutions were organized in an evenly dispersed pattern ( Fig.25 ).

[0322] Sequencing of rolling circle products demonstrates the ability of Phi29 DNA polymerase to reverse transcribe RNA

[0323] Because the amplification rate of Phi29 polymerase is inversely proportional to the number of RNA bases in the substrate, we thought that the enzyme might ignore RNA positions during RCA, thereby introducing single-nucleotide or double-nucleotide deletions in the amplified product. To test this hypothesis, loops containing single and double RNA substitutions (rAr / Ur / G / rC / rArA / rUrU / rGrG / rCrC, Table 9) were amplified as described above and the RCA rate was monitored in real time. After amplification, sequencing libraries were prepared from the different amplified products and then sequenced using Illumina 550 system sequencing. Full-length sequencing reads were extracted from the data set, the sequencing reads were aligned, and the base frequency at each position in the padlock probe backbone was calculated ( Fig. 20 ).

[0324] Based on our observations, Phi29 DNA polymerase incorporated the expected DNA nucleotides in amplified RCPs where the template sequence was RNA. For circles without RNA substitutions, >99% of sequenced monomers showed correctly incorporated bases at the R1 padlock probe region (referred to here as accuracy), which was Fig. 20Highlighted in (99.68%, RT accuracy of position R1 of DNA padlock probe). When R1 position was replaced by rA, rC, rG or rU, RT accuracy was 99.88%, 99.70%, 96.07%, 99.88% respectively. Although rA, rC and rU were copied with better accuracy than sequencing, or at least not worse than dG in the studied position, rG stood out with higher replication error. Interestingly, this higher incorporation error was observed not only for R1 position, but also for all subsequent cytosine bases in the padlock probe backbone. When R1 and R2 positions were replaced by rArA, rCrC, rGrG and rUrU, the RT accuracy of R1 / R2 sites was 99.81 / 99.59%, 99.82 / 99.86%, 93.01 / 89.7% and 99.93 / 99.94% respectively. Similar to the loop with a single rG substitution in R1, all dinucleotide RNA substrates showed higher error rates toward non-RNA cytosines throughout the probe backbone sequence.

[0325] In another experiment, when the R1 position was replaced by rA, rC, rG, or rU, the average error rates were 0.111%, 0.153%, 2.259%, and 0.084%, respectively ( Fig.30 , Fig.31 , Fig.33 ). While rA, rC, and rU were copied with the same accuracy as DNA (as measured by sequencing), rG stood out with higher replication errors. Interestingly, higher incorporation errors were observed not only for the R1 position, but also for all guanosine bases in the padlock probe backbone (in Fig.31 and Fig.33 The high error rate peaks are visible in Fig.30 A higher frequency of thymine was observed in the rG padlock probe signature in . When both R1 and R2 positions were substituted with rArA, rCrC, rGrG, and rUrU, the error rates at the R1 / R2 sites were 0.269 / 0.561%, 0.107 / 0.109%, 2.827 / 2.231%, and 0.144 / 0.220%, respectively. Similar to the loop with a single rG substitution in R1, all dinucleotide RNA substrates showed higher error rates for non-RNA guanosines in the entire probe backbone sequence ( Figure 3 C).

[0326] We demonstrate limited reverse transcription activity of Phi29 DNA polymerase. We confirmed that single RNA substitutions in circular templates had no effect on RCA efficiency. However, we have found that amplification is inhibited when more consecutive RNA bases are substituted in the circular template sequence. To characterize this novel activity of Phi29 polymerase, we amplified circular templates containing one, two or three consecutive RNA bases rA, rG, rC or rU with Phi29 polymerase and monitored the RCA rate in real time. In addition, we tested various combinations of different RNA bases with spacer RNA bases and DNA bases. Our data demonstrate a preference for circular substrates containing pyrimidine RNA bases, as loops with 3 consecutive pyrimidine bases can still be amplified, but loops with 3 consecutive purine bases cannot be amplified. Interestingly, replacing the spacer loop of 3 RNA with DNA bases resulted in a partial recovery of RCA efficiency, indicating that RCA of RNA-containing loops is limited to single RNA base substitutions or very short stretches of consecutive RNA bases. Attempts to improve the efficiency of RCA of circles containing longer stretches of RNA bases by adding reverse transcriptase failed. Instead, RCA was inhibited in the presence of a dedicated reverse transcriptase, presumably due to blocking the circular substrate from binding to the Phi29 DNA polymerase.

[0327] Our data clearly indicate that the mechanism by which the polymerase copies the RNA-containing loop is reverse transcription, as we found that matching DNA bases were incorporated into the RCA products at high frequencies (>99% for rA, rU, and rC, and ~96% for rG). The overall incorporation accuracy of RNA substituents was indistinguishable from that of pure DNA substituents.

[0328] Example 6 - In situ detection of mRNA using chimeric probes

[0329] Materials and methods

[0330] BjHtert and MEF cells were cultured in a growth medium consisting of Dulbecco's modified Eagle's medium (DMEM; Invitrogen), 10% fetal bovine serum (Sigma), and 1% penicillin-streptomycin mixture (PEST; Gibco). Both cell lines were grown in a humidified cell culture incubator at 37°C in the presence of 5% CO2. Prior to the experiment, cells were removed from the culture flask using a 0.25% solution of trypsin-EDTA (T4049 Sigma) and cultured overnight in a 150-mm cell culture dish with 5 immersed microscope slides. The slides with attached cells were washed twice with PBS and fixed on ice in PBS treated with freshly prepared diethylpyrocarbonate (DEPC) containing 3.4% formaldehyde for 15 min. Thereafter, the slides were washed twice with DEPC-PBS, dehydrated with an ethanol gradient (70%, 85%, and 99%; 3 minutes each), air-dried, and stored at 80°C. On the day of the experiment, the cells were thawed, dried, and separated by covering a safety seal chamber (Invitrogen) with an 8 mm diameter and 50 ul volume for each test condition. The 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). All incubations were carried out in a humid chamber to avoid evaporation of the reaction mixture.

[0331] Probes for both ACTB transcripts (Table 4) were combined and prehybridized at a final concentration of 0.1 μM in hybridization buffer (475 mM Tris-HCl at pH 8; 0.95 mM EDTA, 760 mM NaCl. 0.8 U / μL RNA inhibitor (DNAGdansk) shown in 4) in a 50 uL reaction volume at 37°C for 2 hours (Pool 1: non-chimeric PLP, Pool 2: chimeric PLP, Pool 3: iLock, Pool 4: chimeric iLock). Unhybridized probes were removed by rigorous washing twice with preheated (37°C) TBS-Tween buffer. Ligation reactions were performed by adding 0.5 U / μL SplintR ligase (NEB), 1x SplintR buffer, 0.8 U / μL RNase inhibitor in DEPC-ddH2O. iLock ligation and activation (for iLock probes) were performed by simultaneously adding Taq DNA polymerase at a final concentration of 0.1 U / μL. Slides were incubated at 37°C for 2 hours and washed twice with DEPC-PBS-T.

[0332] Rolling circle amplification reactions were performed 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 a final reaction volume of 50 μL at 37°C for 6 h and washed twice with DEPC-PBS-T.

[0333] Finally, the decorated oligonucleotides were hybridized with the RCA products at 0.1 μM final concentration in hybridization buffer (2X SSC, 20% formamide, ddH2O) with Hoechst 33342 (Thermo Scientific) in DEPC-PBS at room temperature for 30 minutes. The cells were washed twice with DEPC-PBS-T, dehydrated through an ethanol series (70, 85 and 99.5% ethanol, 3 min each), and the coverslips were fixed with Slow-Fade medium (Thermo Scientific). The signals in the cells were quantified using CellProfiler software and analyzed in R!

[0334] result

[0335] Despite the incorporation of probes, only the expected signal was observed in cells ( Fig.21 A). In addition, the chimeric padlock probes functioned more efficiently (generated more detectable RCA product) when compared to the non-chimeric padlock probes. The iLock probes generated significantly less signal when compared to the conventional padlock probes, suggesting that further optimization of the protocol is needed to ensure efficient probe activation and in situ RNA detection. However, data analysis revealed that the expected signals were observed for both chimeric and non-chimeric iLock probes, and that the signals for the chimeric iLock probes were also higher ( Fig.21 B).

[0336] Example 7 - Using DNA Detection of PLP, Chimeric PLP, and DNA and Chimeric iLock Probes on Solid Supports miR21.

[0337] In this example, miR21 was immobilized on a glass slide surface and detected in situ. miR21 was prepared by separating the 5' biotin moiety from the target sequence using a 16x rU linker. miR21 was detected using conventional and chimeric padlock probes as well as non-chimeric and chimeric iLock probes. The target and probe sequences are shown in Table 5.

[0338] Materials and methods

[0339] A safety seal chamber (Invitrogen) with a diameter of 8 mm and a volume of 50 ul was placed on a microscope slide (PolyAn) coated with neutral biotin. Six safety seal silicone chambers were used in total. The miR21 target (miR21_BIO) was diluted to a final concentration of 50 nM in a 1x labeling solution (2x SSC, 20% formamide) incubated at room temperature and gently shaken for 1 hour. In one case, the miR21 target (negative control) was intentionally omitted. After miR21 was fixed, the chamber was washed 3 times with PBS-Tween 20 (0.05%). The chamber in which no connection or activation occurred (coating control) was kept in PBS until the end of the experiment.

[0340] The padlock probe, iLock probe, and "coating control" probe (anti-miR21_FAM) were hybridized to the immobilized target at a final concentration of 10 pM (padlock probe and iLock probe) or 50 nM (for anti-miR21_FAM probe). The probes were hybridized in hybridization buffer (475 mM Tris-HCl at pH 8; 0.95 mM EDTA, 760 mM NaCl) at 45°C for 15 minutes and gently shaken at room temperature for 3 hours. The chamber was then washed twice with PBS-Tween 20 (0.05%).

[0341] Ligation reaction was performed by adding 0.5U / uL SplintR ligase (NEB), 1x SplintR buffer in DEPC-ddH2O. iLock ligation and activation (for iLock probes) were performed by adding Taq DNA polymerase at a final concentration of 0.1U / uL simultaneously. Slides were incubated at 37°C for 1 hour and washed twice with DEPC-PBS-T.

[0342] Rolling circle amplification reactions were performed 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 a final reaction volume of 50 μL at room temperature for 3 hours and washed twice with DEPC-PBS-T.

[0343] Finally, the decorated oligonucleotides were hybridized with the RCA products at a final concentration of 0.1 μM in hybridization buffer (2X SSC, 20% formamide, ddH2O) in DEPC-PBS at room temperature for 1 hour. The cells were washed twice with DEPC-PBS-T, dehydrated in 99% ethanol for 3 min, and the coverslips were fixed with Slow-Fade medium (Thermo Scientific). The signals in the cells were quantified using CellProfiler software.

[0344] result

[0345] Our data indicate that biotinylated miRNA targets were effectively immobilized on neutral biotin-coated microscope slides because no fluorescence was detected from the labeled complementary probe when miR21 was not immobilized. Fig. 22 B. The conventional padlock probe produced approximately 7800 RCA products (RCPs) / field of view (fov), while approximately 36000 RCPs / fov were quantified when the chimeric padlock probe was used. Consistent with the example in which ACTB mRNA was detected in BjhTERT and MEF cultured cells, the iLock probe produced less signal compared to the padlock probe. The chimeric iLock probe produced approximately 3000 RCPs / fov, while the unmodified iLock probe produced only approximately 195 RCPs / fov.

[0346] Example 8 - In situ multiplexed gene expression profiling in mouse brain tissue sections using chimeric padlock probes and cell type analysis and in situ sequencing

[0347] Materials and methods

[0348] Immediately after surgical resection and without any fixation, P30 mouse brain is embedded in OCT culture medium and directly frozen on dry ice, and thereafter stored at -80 ℃ until use. Then 10 μm slices are cut with a cryostat and the slices are collected on Superfrost glass slides. Then the slices are briefly fixed for 5min at room temperature in 3.7% PFA in DEPC-treated PBS. Afterwards, the slices are washed once in 0.05% DEPC-PBS Tween and permeabilized with 0.1M HCl at room temperature for 5min. After permeabilization, the slides are washed twice in DEPC-PBS and dehydrated for 2min by 70%, 85% and 100% ethanol series respectively. The safety seal chamber is mounted on the slide covering the tissue slice, and the tissue is hydrated by briefly washing with PBS-T (DEPC-PBS containing 0.05% Tween). In order to target mRNA with chimeric padlock probe (PLP), after short rehydration washing, the slices were immersed in a chimeric PLP hybridization mixture containing 2x SSC buffer, 20% formamide, 0.05M KCl, 0.2mg / mL BSA, 1U / μL RNase inhibitor and 50nM chimeric PLP. Hybridization was carried out overnight at 45°C. Afterwards, the slices were washed 15min at 37°C in preheated buffer (2x SSC, 20% formamide). Finally, the slices were washed 2 times in PBS-T. Then a ligation reaction mixture was added to the slices, and the ligation reaction mixture contained 1x SplintR ligase buffer, 0.2mg / mL BSA, 0.8U / μL RNase inhibitor and 0.25U / μL SplintR ligase. The ligation reaction was incubated at 37°C for 60min. The slices were washed twice in PBS-T. Next, the sections were immersed in a rolling circle amplification mixture containing 1x phi29 polymerase buffer, 0.25 mM dNTPs, 0.2 mg / mL BSA, 1 U / μL phi29 polymerase, 5% glycerol, and 50 nM RCA primers. RCA was performed at 37°C for 3 h. Subsequently, the sections were washed twice in PBS-T and the detection probe (acting as an anchor probe in the in situ sequencing reaction) was hybridized in situ with the RCA product in 2x SSC and 20% formamide hybridization buffer at room temperature for 30 min.

[0349] For in situ sequencing, the sections were immersed in a ligation sequencing mixture containing 1x T4 ligation buffer, 1mM ATP, 0.2mg / mL BSA, 0.1U / μL T4 DNA ligase, and 100nM of each sequencing library base 1 (for sequencing the first barcode position, for sequencing the second barcode position, etc.) as previously described in Ke et al. (2013, Nature methods). The sequencing reaction was incubated at room temperature for 1h. The sections were then washed 3 times in PBS-T and the nuclei were stained with DAPI, washed three times again, a short ethanol series was performed as described above, and the tissue was then fixed in Slowfade mounting medium. The tissue sections were then imaged in a fluorescence microscope with a 20x objective. To sequence the second base, the sections were first washed in ethanol to remove the mounting medium, and then washed twice in 100% formamide to strip the anchor probe and the connected sequencing probe. The sections were washed 3 times in PBS-T and then the ligation sequencing mix for the second base (same composition as above) was added to the sections and the procedure was repeated for positions 3 and 4. The images of the sequencing reactions were then processed by Cell profiler software and Matlab scripts as previously described in Ke et al. (Nat methods 2013).

[0350] result

[0351] The use of cDNA synthesis for multiplexed in situ gene expression profiling and subsequent targeting of cDNA by DNA padlock probes (PLPs) is generally limited to highly expressed genes due to the low efficiency of cDNA synthesis. Direct targeting of RNA with PLPs has been difficult to date due to low efficiency of probe ligation of RNA by the enzyme and insufficient specificity leading to false positive signals. In this experiment, we demonstrated that chimeric PLPs ligate RNA very efficiently ( Fig.23). We applied chimeric PLPs targeting 18 different genes on mouse brain tissue sections, 5 probes per gene (90 probes in total) (Table 10). The probes were barcoded with sequencing barcodes that could be decoded later by in situ sequencing. The probes were first hybridized to RNA and then ligated using SplintR ligase after washing. The use of T4 RNA ligase 2 can further improve specificity, as we have demonstrated that specificity and efficiency are improved using T4 RNA ligase 2 (see previous examples). The ligated probes were amplified with RCA and the barcodes in the RCA products were sequenced by ligation chemistry sequencing, as previously described in Ke et al. (Nat methods 2013). The overall expression pattern obtained using the direct RNA method of chimeric PLPs is very comparable to that obtained by traditional cDNA targeting methods (data not shown). For simplicity, general staining of all RCA products is presented in this example. In addition to the advantage of high sensitivity, the chimeric PLP direct RNA method has a lower assay cost because the cDNA synthesis step is associated with the high cost of reverse transcriptase, and the assay can be performed faster because the cDNA synthesis step is omitted. Overall, the chimeric probes show potential for highly multiplexed RNA analysis in combination with in situ sequencing readouts in tissue sections.

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[0372] Sequence Listing <110> 10X Genomics Ltd. <120> RNA-templated ligation <130> 27.35.133725 / 03 <150> GB 1716407.0 <151> 2017-10-06 <150> GB 1718095.1 <151> 2017-11-01 <150> GB 1718089.5 <151> 2017-11-01 <160> 281 <170> PatentIn version 3.5 <210> 1 <211> 41 <212> RNA <213> Homo sapiens <400> 1 aacuugggu aguuggagcu gguggcguag gcaagagugc c 41 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> KRASwt_PLP <220> <221> misc_feature <222> (1) <223> 5' phosphate <400> 2 agctccaact accacaaaaa aaaaaagtag ccgtgactat cgactaaaaa aaaaacttgc 60 ctacgccacc 70 <210> 3 <211> twenty two <212> RNA <213> Homo sapiens <400> 3 ugagguagga gguuguauag uu 22 <210> 4 <211> twenty two <212> RNA <213> Homo sapiens <400> 4 agagguagua gguugcauag uu 22 <210> 5 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-a_PLP_1 <220> <221> misc_feature <222> (1) <223> 5' Phosphate <400> 5 ctactacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaac 65 <210> 6 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-f_PLP_1 <220> <221> misc_feature <222> (1) <223> 5' Phosphate <400> 6 ctactacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaat 65 <210> 7 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-e_PLP_1 <220> <221> misc_feature <222> (1) <223> 5' Phosphate <400> 7 ctcctacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaac 65 <210> 8 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-d_PLP_1 <220> <221> misc_feature <222> (1) <223> 5' phosphate <400> 8 ctactacctc taaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 GCAAC 65 <210> 9 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-a_PLP_RNA_1 <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 9 ctactacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaac 65 <210> 10 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-f_PLP_RNA_1 <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 10 ctactacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaau 65 <210> 11 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-e_PLP_RNA_1 <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 11 ctcctacctc aaaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 acaac 65 <210> 12 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> let7-d_PLP_RNA_1 <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 12 ctactacctc taaaaaaaaa acctcaatgc acatgtttgg ctccaaaaaa aaaaaactat 60 GCAAC 65 <210> 13 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_C <400> 13 ucucgcuguc aucccuauau ccucg 25 <210> 14 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_A <400> 14 ucucgcuguc auaccuauau ccucg 25 <210> 15 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_G <400> 15 ucucgcuguc augccuauau ccucg 25 <210> 16 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_U <400> 16 ucucgcuguc auuccuauau ccucg 25 <210> 17 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> 3'T_PLP_2 <220> <221> misc_feature <222> (1) <223> 5' Phosphate <400> 17 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata 60 taggt 65 <210> 18 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> 3'G_PLP_2 <220> <221> misc_feature <222> (1) <223> 5' phosphate <400> 18 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata 60 taggg 65 <210> 19 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> 3'A_PLP_2 <220> <221> misc_feature <222> (1) <223> 5' phosphate <400> 19 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata 65 tags <210> 20 <211> 65 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'C_PLP_2 <220> <221> misc_feature <222> (1)..(1) <223> 5'shell <400> 20 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata tagc 65 <210> 21 <211> 65 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'rT_PLP_2 <220> <221> misc_feature <222> (1)..(1) <223> 5'shell <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 21 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata labeled <210> 22 <211> 65 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'rG_PLP_2 <220> <221> misc_feature <222> (1)..(1) <223> 5'shell <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 22 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata taggg <210> 23 <211> 65 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'rA_PLP_2 <220> <221> misc_feature <222> (1)..(1) <223> 5'shell <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> 23 atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata 65 tags <210> 24 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> 3'rC_PLP_2 <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (65)..(65) <223> RNA <400> twenty four atgacagcga gaaaaaaaaa aaagtagccg tgactatcga ctaaaaaaaa aacgaggata 60 taggc 65 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Decorative probe_1 <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (21)..(24) <223> 2' O-Me RNA <400> 25 cctcaatgca catgtttggc tcca 24 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Decorative probe 2_2 <220> <221> misc_feature <222> (1)..(1) <223> 5'shell <220> <221> misc_feature <222> (18)..(21) <223> 2' O-Me RNA <400> 26 holds a 21 actatcgact <210> 27 <211> 22 <212> RNA <213> Man(Homo sapiens) <400> 27 22. snow snow snow snow <210> 28 <211> 59 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> let-7a_PLP <400> 28 ctactacctc aaaaaaacc tcaatgcaca tgtttggctc caaaaaaaaa ctatacaac <210> 29 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock_1 <400> 29 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac <210> 30 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock-3_1 <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 30 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac <210> 31 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock-3D_1 <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 31 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac <210> 32 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock-3D5_1 <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 32 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac <210> 33 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock-3DF_1 <220> <221> misc_feature <222> (1)..(14) <223> RNA <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 33 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac <210> 34 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> iLock-DF_1 <220> <221> misc_feature <222> (1)..(14) <223> RNA <400> 34 cgcgtgtcgt tgccctacta cctcaaaaaaa aaaaacctca atgcacatgt ttggctccaa aaaaaaaaaa ctatacaac 79 <210> 35 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_C <400> 35 ucucgcuguc aucccuauau ccucg 25 <210> 36 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_A <400> 36 ucucgcuguc auaccuauau ccucg 25 <210> 37 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_G <400> 37 ucucgcuguc augccuauau ccucg 25 <210> 38 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> benchm_templ_U <400> 38 ucucgcuguc auuccuauau ccucg 25 <210> 39 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'T_iLock_3 <400> 39 60. tatatcccta tattatgaca gcgagaaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag throw <210> 40 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'G_iLock_3 <400> 40 60. tatatcccta tatgatgaca gcgagaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag 79 <210> 41 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'A_iLock_3 <400> 41 60. tatatcccta tatatgaca gcgagaaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag 79 years old <210> 42 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'C_iLock_3 <400> 42 60. tatatcccta tatcatgaca gcgagaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag throw <210> 43 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'U_iLock_RNA_3 <220> <221> misc_feature <222> (13)..(13) <223> RNA <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 43 60. tatatcccta tatuatgaca gcgagaaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag 79th floor <210> 44 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'G_iLock_RNA_3 <220> <221> misc_feature <222> (13)..(13) <223> RNA <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 44 60. tatatcccta tatgatgaca gcgagaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag 79 <210> 45 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'A_iLock _RNA_3 <220> <221> misc_feature <222> (13)..(13) <223> RNA <220> <221> misc_feature <222> (79)..(79) <223> RNA <400> 45 60. tatatcccta tatatgaca gcgagaaaaaa aaaaaaagta gccgtgacta tcgactaaaa aaaaaacgag 79 years old <210> 46 <211> 79 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> 3'C_iLock _RNA_3 <220> <221> misc_feature <222> (13)..(13) <220> <221> misc_feature <222> (79)..(79) <400> 46 tatatcccta tatcatgaca gcgagaaaaa aaaaaaagta gccgtgacta tcgactaaaa 60 aaaaaacgag gatataggc 79 <210> 47 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Decorative probe_1 <400> 47 cctcaatgca catgtttggc tcca 24 <210> 48 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Decorative probe_2 <400> 48 tgcgtctatt tagtggagcc a 21 <210> 49 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Decorative probe_3 <400> 49 agtagccgtg actatcgact a 21 <210> 50 <211> twenty two <212> RNA <213> Homo sapiens <400> 50 ugagguagua gguuguauag uu 22 <210> 51 <211> twenty two <212> RNA <213> Homo sapiens <400> 51 ugagguagua gauuguauag uu 22 <210> 52 <211> twenty two <212> RNA <213> Homo sapiens <400> 52 ugagguagga gguuguauag uu 22 <210> 53 <211> twenty two <212> RNA <213> Homo sapiens <400> 53 agagguagua gguugcauag uu 22 <210> 54 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7a_seqRNA <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (69)..(69) <223> RNA <400> 54 aaagatgcga tacactacct catgcgtcta tttagtggag cccgctatct tctttaacta 60 tacaaccta 69 <210> 55 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7e_seqRNA <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (69)..(69) <223> RNA <400> 55 aaaatgtcgt tgccctccta cctcatgcgt ctatttagtg gagccgccta tcttctttaa ctatacaac <210> 56 <211> 69 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> let-7d_seqRNA <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (69)..(69) <223> RNA <400> 56 aaaatgtcgt tgccctacta cctcttgcgt ctatttagtg gagccatcta tcttctttaa 69. ctatgcaac <210> 57 <211> 69 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> let-7f_seqRNA <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (69)..(69) <223> RNA <400> 57 aaaatgtcgt tgcuctacta cctcatgcgt ctatttagtg gagcctacta tcttttaa 60 ctatacaau 69 <210> 58 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7a_seq <400> 58 aaagatgcga tacactacct catgcgtcta tttagtggag cccgctatct tctttaacta 60 tacaacta 69 <210> 59 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7e_seq <400> 59 aaaatgtcgt tgccctccta cctcatgcgt ctatttagtg gagccgccta tcttctttaa 60 ctatacaac 69 <210> 60 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7d_seq <400> 60 aaaatgtcgt tgccctacta cctcttgcgt ctatttagtg gagccatcta tcttctttaa 60 ctatgcaac 69 <210> 61 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> let-7f_seq <400> 61 aaaatgtcgt tgctctacta cctcatgcgt ctatttagtg gagcctacta tcttctttaa 60 ctatacaat 69 <210> 62 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Decorative probe <220> <221> misc_feature <222> (1) <223> 5' Cy3 <400> 62 tgcgtctatt tagtggagcc a 21 <210> 63 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Anchor Primer <220> <221> misc_feature <222> (1) <223> 5' AlexaFluor750 <400> 63 tgcgtctatt tagtggagcc a 21 <210> 64 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> seqlibb1T <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (2)..(9) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' FITCb <400> 64 tnnnnnnnn 9 <210> 65 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> seqlibb1G <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (2)..(9) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy3b <400> 65 gnnnnnnnn 9 <210> 66 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> seqlibb1A <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (2)..(9) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5b <400> 66 annnnnnnnn 9 <210> 67 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> seqlibb1C <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (2)..(9) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' TexasRedb <400> 67 cnnnnnnnn 9 <210> 68 <211> 54 <212> DNA <213> House mouse (Mus musculus) <400> 68 ggcctgtaca ctgacttgag accaataaaa gtgcacacct taccttacac aaac 54 <210> 69 <211> 94 <212> DNA <213> Homo sapiens <400> 69 tacctgtaca ctgacttgag accagttgaa taaaagtgca caccttaaaa atgaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 94 <210> 70 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Mouse PLP <400> 70 taaggtgtgc aaaaagtagc cgtgactatc gactaaaagt agccgtgact atcgactgtt 60 tgtgtaagg 69 <210> 71 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Human PLP <400> 71 taaggtgtgc aagtcggaag tactactctc taaaagtcgg aagtactact ctcttttttt 60 ttcatttt 68 <210> 72 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Mouse cPLP <220> <221> misc_feature <222> (69)..(69) <223> RNA <400> 72 taaggtgtgc aaaaagtagc cgtgactatc gactaaaagt agccgtgact atcgactgtt 60 tgtgtaagg 69 <210> 73 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Human cPLP <220> <221> misc_feature <222> (68)..(68) <223> RNA <400> 73 taaggtgtgc aagtcggaag tactactctc taaaagtcgg aagtactact ctcttttttt 60 ttcatttu 68 <210> 74 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Also iLock <400> 74 fathercccta father gtgcaaaaag father father aagtagccgt gactatcgac tgtttgtgta agg <210> 75 <211> 82 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Remove iLock <400> 75 fathercccta father gtgcaagtcg gaagtac tctctaaaag tcggaagtac tactctcttt ttttttcatt tt 82 <210> 76 <211> 83 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Free ciLock <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (83)..(83) <223> RNA <400> 76 fathercccta father gtgcaaaaag father father aagtagccgt gactatcgac tgtttgtgta agg <210> 77 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Human ciLock <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (82)..(82) <223> RNA <400> 77 tatatcccta tatutaaggt gtgcaagtcg gaagtactac tctctaaaag tcggaagtac 60 tactctcttt ttttttcatt tu 82 <210> 78 <211> 37 <212> RNA <213> Artificial Sequence <220> <223> miR21_BIO <400> 78 uuuuuuuuuu uuuuuuagcu uaucagacug auguuga 37 <210> 79 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> >miR21B2DO_PLP <220> <221> misc_feature <222> (1) <223> 5' Phosphate <400> 79 ctgataagct agccgaatct aagagtagcc gtgactatcg actaaaacta cacca 55 <210> 80 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> >miR21B2DO_iLock <400> 80 ccgtcgctgc gttctgataa gctaaaaaaa agtagccgtg actatcgact aaaaaaatca 60 acatcagt 68 <210> 81 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> > miR21 B2DO_rPLP <220> <221> misc_feature <222> (1)..(1) <223> 5'-phosphate <220> <221> misc_feature <222> (1)..(1) <223> phosphate <220> <221> misc_feature <222> (66)..(66) <223> RNA <400> 81 ctgataagct agccgaatct aagagtagcc gtgactatcg actaaaacta caccatcaac 60 atcagu 66 <210> 82 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> >miR21B2DO_RiLock <220> <221> misc_feature <222> (13) <223> RNA <220> <221> misc_feature <222> (68)..(68) <223> RNA <400> 82 ccgtcgctgc gtuctgataa gctaaaaaaa agtagccgtg actatcgact aaaaaaatca 60 acatcagu 68 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> >B2DO_Cy3 <220> <221> misc_feature <222> (1) <223> 5' Cy3 <400> 83 agtagccgtg actatcgact 20 <210> 84 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> >antimiR21_FAM <220> <221> misc_feature <222> (1) <223> 5' FAM <400> 84 tcaacatcag tctgataagc ta <210> 85 <211> 99 <212> DNA <213> Man(Homo sapiens) <400> 85 acattttcat tatttttatt ataaggcctg ctgaaaatga ctgaatataa acttgtggta gttggagctg gtggcgtagg caagagtgcc ttgacgata <210> 86 <211> 77 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> wt CROSS PLP <400> 86 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg cccttgccta cgccacc 77 <210> 87 <211> 41 <212> RNA <213> Man(Homo sapiens) <400> 87 aacuuguggu aguuggagcu gguggcguag gcaagagugc c <210> 88 <211> 77 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> chim wt KRAS PLP <220> <221> misc_feature <222> (77)..(77) <223> RNA <400> 88 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 89 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> chim wt KRAS PLP_2 <220> <221> misc_feature <222> (76)..(77) <223> RNA <400> 89 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 90 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> mut KRAS PLP <400> 90 agctccaact accacaacct caatgcacat gtttggctcc cagacgtaac gcgttcagtg 60 atgcccttgc ctacgccact 80 <210> 91 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> chim mut KRAS PLP <220> <221> misc_feature <222> (80)..(80) <223> RNA <400> 91 agctccaact accacaacct caatgcacat gtttggctcc cagacgtaac gcgttcagtg 60 atgcccttgc ctacgccacu 80 <210> 92 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> RCA Primers <400> 92 catcactgaa cgcgt 15 <210> 93 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> mut DO-Cy5 <400> 93 cctcaatgca catgtttggc tcc 23 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> wt DO-Cy3 <400> 94 agtcgatagt cacggctact 20 <210> 95 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> KRAS wt invasion chim 3-5 <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (91)..(91) <223> RNA <400> 95 tatatcccta tatcagctcc aactaccaca aagtcgatag tcacggctac tcagacgtaa 60 cgcgttcagt gatgcccttg cctacgccac c 91 <210> 96 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> KRAS wt invasion chim 3 <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (91)..(91) <223> RNA <400> 96 tatatcccta tatcagctcc aactaccaca aagtcgatag tcacggctac tcagacgtaa 60 cgcgttcagt gatgcccttg cctacgccac c 91 <210> 97 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> KRAS mut invasion chim 3-5 <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (94)..(94) <223> RNA <400> 97 tatatcccta tatuagctcc aactaccaca acctcaatgc acatgtttgg ctcccagacg 60 taacgcgttc agtgatgccc ttgcctacgc cacu 94 <210> 98 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> KRAS mut invasion chim 3 <220> <221> misc_feature <222> (14)..(14) <223> RNA <220> <221> misc_feature <222> (94)..(94) <223> RNA <400> 98 tatatcccta tattagctcc aactaccaca acctcaatgc acatgtttgg ctcccagacg 60 taacgcgttc agtgatgccc ttgcctacgc cacu 94 <210> 99 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> KRAS gap-filling invaders <400> 99 tatatctcta tatagctcca actaccacaa gtagtcgata gtcacggcta ctcagacgta 60 acgcgttcag tgatgagtag gcactcttgc ctac 94 <210> 100 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Intrusion_splint_chim <220> <221> misc_feature <222> (19)..(19) <223> RNA <400> 100 tatatctcta tatgccacc 19 <210> 101 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Intrusion_Splint <400> 101 tatatctcta tatgccacc 19 <210> 102 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Splint <220> <221> misc_feature <222> (6) <223> RNA <400> 102 gccacc 6 <210> 103 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Splint_chim <400> 103 gccacc 6 <210> 104 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> RCA Primers <400> 104 catcactgaa cgcgt 15 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DO-Cy3 <400> 105 agtcgatagt cacggctact 20 <210> 106 <211> 41 <212> RNA <213> Homo sapiens <400> 106 aacuugggu aguuggagcu gguggcguag gcaagagugc c 41 <210> 107 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_1 <220> <221> misc_feature <222> (20)..(20) <223> RNA <400> 107 agctccaact accacaacag acgtaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 108 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_2 <220> <221> misc_feature <222> (20)..(21) <223> RNA <220> <221> misc_feature <222> (29)..(30) <223> RNA <400> 108 agctccaact accacaacag acgtaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 109 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_3 <220> <221> misc_feature <222> (20)..(22) <223> RNA <220> <221> misc_feature <222> (29)..(30) <223> RNA <400> 109 agctccaact accacaacag acgtaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 110 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_4 <220> <221> misc_feature <222> (20)..(23) <223> RNA <220> <221> misc_feature <222> (29)..(30) <223> RNA <400> 110 agctccaact accacaacag acgtaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 111 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_5 <220> <221> misc_feature <222> (20)..(24) <223> RNA <220> <221> misc_feature <222> (29)..(30) <223> RNA <400> 111 agctccaact accacaacag acguaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 112 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_7 <220> <221> misc_feature <222> (20)..(26) <223> RNA <220> <221> misc_feature <222> (29)..(30) <223> RNA <400> 112 agctccaact accacaacag acguaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 113 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_6S <220> <221> misc_feature <222> (20)..(21) <223> RNA <220> <221> misc_feature <222> (24)..(25) <223> RNA <220> <221> misc_feature <222> (28)..(29) <223> RNA <400> 113 agctccaact accacaacag acguaacgcg ttcagtgatg cccttgccta cgccacc 57 <210> 114 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_DNA <400> 114 agctccaact accacaaaaa aaaaaaaaaa aaaaacttgc ctacgccacc 50 <210> 115 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS wt1_RNA <220> <221> misc_feature <222> (50)..(50) <223> RNA <400> 115 agctccaact accacaaaaa aaaaaaaaaa aaaaacttgc ctacgccacc 50 <210> 116 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Decorative probe[1] <220> <221> misc_feature <222> (19)..(22) <223> 2' O-Me RNA <400> 116 cctcaatgca catgtttggc tc 22 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Decorative Probe 2 [2] <220> <221> misc_feature <222> (17)..(20) <223> 2' O-Me RNA <400> 117 agtcgatagt cacggctact 20 <210> 118 <211> 41 <212> RNA <213> Homo sapiens <400> 118 aacuugggu aguuggagcu gguggcguag gcaagagugc c 41 <210> 119 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.0 <400> 119 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 120 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.1G <220> <221> misc_feature <222> (40)..(40) <223> RNA <400> 120 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 121 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.1C <220> <221> misc_feature <222> (40)..(40) <223> RNA <400> 121 agctccaact accacaaagt cgatagtcac ggctactcac acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 122 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.1A <220> <221> misc_feature <222> (40)..(40) <223> RNA <400> 122 agctccaact accacaaagt cgatagtcac ggctactcaa acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 123 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.1U <220> <221> misc_feature <222> (40)..(40) <223> RNA <400> 123 agctccaact accacaaagt cgatagtcac ggctactcau acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 124 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.2G <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 124 agctccaact accacaaagt cgatagtcac ggctactcag gcgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 125 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.2C <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 125 agctccaact accacaaagt cgatagtcac ggctactcac ccgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 126 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.2A <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 126 agctccaact accacaaagt cgatagtcac ggctactcaa acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 127 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.2U <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 127 agctccaact accacaaagt cgatagtcac ggctactcau ucgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 128 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> AluI KRAS RO <400> 128 cgccaccagc tccaacta 18 <210> 129 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> PE1 <400> 129 acactctttc cctacacgac gctcttccga tctctggtgg cgtaggcaag ggc 53 <210> 130 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> PE2 <400> 130 gtgactggag ttcagacgtg tgctcttccg atctctccaa ctaccacaaa gtcg 54 <210> 131 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3G <220> <221> misc_feature <222> (40)..(42) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 131 agctccaact accacaaagt cgatagtcac ggctactcag ggttaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 132 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3C <220> <221> misc_feature <222> (40)..(42) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 132 agctccaact accacaaagt cgatagtcac ggctactcac ccgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 133 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3A <220> <221> misc_feature <222> (40)..(42) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 133 agctccaact accacaaagt cgatagtcac ggctactcaa aagtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 134 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3U <220> <221> misc_feature <222> (40)..(42) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 134 agctccaact accacaaagt cgatagtcac ggctactcau uucgtaacgc gttcagtgat 60 gcccttgcct acgccacc 78 <210> 135 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.5 <220> <221> misc_feature <222> (40)..(44) <223> RNA <400> 135 agctccaact accacaaagt cgatagtcac ggctactcag acguaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 136 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.6S <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (44)..(45) <223> RNA <220> <221> misc_feature <222> (48)..(49) <223> RNA <400> 136 agctccaact accacaaagt cgatagtcac ggctactcag acguaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 137 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3 <220> <221> misc_feature <222> (40)..(42) <223> RNA <220> <221> misc_feature <222> (48)..(50) <223> RNA <400> 137 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 138 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.3S <220> <221> misc_feature <222> (40)..(40) <220> <221> misc_feature <222> (42)..(42) <220> <221> misc_feature <222> (44)..(44) <400> 138 agctccaact accacaaagt cgatagtcac ggctactcag acguaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 139 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.2 <220> <221> misc_feature <222> (40)..(41) <223> RNA <220> <221> misc_feature <222> (49)..(50) <223> RNA <400> 139 agctccaact accacaaagt cgatagtcac ggctactcag acgtaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 140 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> R_KRAS.7 <220> <221> misc_feature <222> (40)..(46) <223> RNA <400> 140 agctccaact accacaaagt cgatagtcac ggctactcag acguaacgcg ttcagtgatg 60 cccttgccta cgccacc 77 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Detection probe (sequence anchor probe) <220> <221> misc_feature <222> (1) <223> 5' AlexaFluor750 <400> 141 tgcgtctatt tagtggagcc 20 <210> 142 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 1st base-G <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (2)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy3 <400> 142 gnnnctatc 9 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 1st base-G <220> <221> misc_feature <222> (20)..(20) <223> 3'-phosphorothioate <400> 143 ggctccacta aatagacgca 20 <210> 144 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 1st base-A <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (2)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5 <400> 144 annnctatc 9 <210> 145 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 1st base-C <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (2)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' TexasRed <400> 145 cnnnctatc 9 <210> 146 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 1st base-T <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (2)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Alexa488 <400> 146 tnnnctatc 9 <210> 147 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 2nd base-G <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (1) <223> n is any nucleotide <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy3 <400> 147 ngnnctatc 9 <210> 148 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 2nd base-A <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1) <223> n is any nucleotide <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5 <400> 148 nannctatc 9 <210> 149 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 2nd base-C <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1) <223> n is any nucleotide <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' TexasRed <400> 149 ncnnctatc 9 <210> 150 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 2nd base-T <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1) <223> n is any nucleotide <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Alexa488 <400> 150 ntnnctatc 9 <210> 151 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 3rd base-G <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(2) <223> n is any nucleotide <220> <221> misc_feature <222> (4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy3 <400> 151 nngnctatc 9 <210> 152 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 3rd base-A <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(2) <223> n is any nucleotide <220> <221> misc_feature <222> (4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5 <400> 152 nnanctatc 9 <210> 153 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 3rd base-C <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(2) <223> n is any nucleotide <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5 <220> <221> misc_feature <222> (9)..(9) <223> 3' TexasRed <400> 153 nncnctatc 9 <210> 154 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 3rd base-T <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1) <223> n is any nucleotide <220> <221> misc_feature <222> (2)..(2) <223> n is a, c, g, or t <220> <221> misc_feature <222> (3)..(4) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Alexa488 <400> 154 nntnctatc 9 <210> 155 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 4th base-G <220> <221> misc_feature <222> (1) <223> 5' phosphate <220> <221> misc_feature <222> (1)..(3) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy3 <400> 155 nnngctatc 9 <210> 156 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 4th base-A <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(3) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Cy5 <400> 156 nnnactatc 9 <210> 157 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 4th base-C <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(3) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' TexasRed <400> 157 nnncctatc 9 <210> 158 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Sequence library 4th base-T <220> <221> misc_feature <222> (1) <223> 5' Phosphate <220> <221> misc_feature <222> (1)..(3) <223> n is any nucleotide <220> <221> misc_feature <222> (9)..(9) <223> 3' Alexa488 <400> 158 nnntctatc <210> 159 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Calb2_1119_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 159 60. catcgcagcg gagacgacag ctgattcctt tgactcacat tgcgtctatt tagtggagcc aatgctatct tctttataca gcgaaggaac tcatg <210> 160 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Calb2_1328_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 160 60. cacacacgtc aagaacacaa ctgattcctt tgactcacat tgcgtctatt tagtggagcc aatgctatct tctttgaagc 95-year-old <210> 161 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Calb2_164_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 161 accttcaatg tacccatttc ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aatgctatct tctttaagaa gttctctagc tcttu 95 <210> 162 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Calb2_500_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 162 aggttcatca tagggcctgt ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aatgctatct tcttttgggt gtactcctgg agctu 95 <210> 163 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Calb2_937_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 163 gtcaagagag tcaggacagc ctgattcctt tgactcacat tgcgtctatt tagtggagcc aatgctatct tctttgggga ggtctgggga ggagu <210> 164 <211> 93 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Calb2_DO3_CCAA_RNA <220> <221> misc_feature <222> (93)..(93) <223> RNA <400> 164 ctcatacaga tccttcagct gattcctttg actcacattg cgtctattta gtggagcccc aactatcttc tttttcatct ccttcttgtt ctu 93 <210> 165 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Chodl_1164_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 165 cgggctagtt tttgatcttc ctgattcctt tgactcacat tgcgtctatt tagtggagcc stable tctttatcca stable tgatu <210> 166 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Chodl_1798_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 166 aaagcaaaga aacagaacaa ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 agtactatct tcttttccta aactttatcg aaccc 95 <210> 167 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Chodl_2071_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 167 attctatagg caacatgtga ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 agtactatct tctttactct ggggagctat ttgca 95 <210> 168 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Chodl_2252_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 168 gttctgctta gcatcacact ctgattcctt tgactcacat tgcgtctatt tagtggagcc agtactatct tctttttaat cattaatc agtgu <210> 169 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Chodl_293_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 169 tcctactccc tccttcccag ctgattcctt tgactcacat tgcgtctatt tagtggagcc agtactatct tctttttcct tgctttcctg ctggg 95 <210> 170 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Chodl_789_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 170 gaactgggag ctgcttccat ctgattcctt tgactcacat tgcgtctatt tagtggagcc agtactatct tcttttcatc agtgtaccag tttcg 95 <210> 171 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Chodl_916_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 171 tgttgcacct gtcgtcattc ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 agtactatct tctttgcaga tgtaattgtg cttca 95 <210> 172 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Cort_326_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 172 cccgggggta ccccctccga cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tgatctatct tctttctttc ctggctcttg gacag 95 <210> 173 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Cort_529_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 173 caaagctgac ataagaagaa cgtgcttgtg gtagcaata tgcgtctatt tagtggagcc tgatctatct tctttttctc acagggcagg ggagg <210> 174 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_1014_GGAC_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 174 agtgtgtctg acacgatgat cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc ggacctatct tcttttaccg atggccgttg ctggc 95 <210> 175 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_1309_GTAA_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 175 atggctgcag tggtttccca cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc gtaacttct tctttaagtc ctgaggtcct ccaac <210> 176 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_1573_TAC_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 176 60. ccaaatggac cagagagtga cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc taacctatct tctttgtgcc cactcaaga taatg <210> 177 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_1750_TATT_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 177 60. aagtcagaga gacagactgg cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc tattctatct tctttgcttt aaagccatga taggg <210> 178 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Htr3a_1927_TCGC_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 178 gcaagacaat ttgcttttct cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tcgcctatct tctttcagaa gtctcaggca tctau 95 <210> 179 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Htr3a_2045_TGGA_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 179 attatcccct gctcccattg cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tggactatct tctttttaag atatcatagc atttu 95 <210> 180 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Htr3a_21_AAGT_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 180 gtcccaggca gactgctttt cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc aagtctatct tctttccacc cgctgccaac ctcau <210> 181 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_247_CAGC_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 181 gtctgacagc cttagtagg cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc cagcctatct tctttttgta gttagccagg aggtg <210> 182 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_424_CGGT_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 182 agtccactgc agaaactcat cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc cggtctatct tctttacatt gtcgaagtcc tcagg 95 <210> 183 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_89_CACT_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 183 ctcagagcag ccactcagga cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc cactctatct tctttcttcc cagatgtggg agggc 95 <210> 184 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Htr3a_955_GCTC_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 184 agagactctc tcaccgctgt cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc gctcctatct tctttagaag gagtgtgatc ttgaa <210> 185 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_1033_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 185 tagaaggatt catatgcact ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttactca ggggaggtac tttca 95 <210> 186 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_108_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 186 ctgctagtga cgtcacaggg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttagagc tggtacccat gccau 95 <210> 187 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_1524_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 187 tgtgatacag acaagaggga ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttagaga gagagagaat cacag 95 <210> 188 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_168_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 188 tctcattgat ctctaaaaag ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttatctg tgtgtatctg cacta 95 <210> 189 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_1688_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 189 agacattgaa gtatgctgtg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttcataa ctgtacaact gaaau 95 <210> 190 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_2041_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 190 aacaatacaa aacaagtgct ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttacctg tacagaaaaa tcctg 95 <210> 191 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_228_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 191 ttttcaggct gagtgtcgca ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttcattt tggatcttcc aaatc 95 <210> 192 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_315_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 192 gtagtgttaa catggttctt ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tcttttacga cagactcgtc aaacg 95 <210> 193 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_495_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 193 tgtcttcttc ctcctcttct ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttattct catcttcttc ctctc 95 <210> 194 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_648_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 194 tgtccagagc atcattgagg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttgggga ccacttttcg caaau 95 <210> 195 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_714_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 195 gtcgtaaagt ttctattttg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 ttgcctatct tctttccaga tgtaattttt ggcca 95 <210> 196 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Neurod6_971_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 196 cccatgccct gggggagtgg ctgattcctt tgactcacat tgcgtctatt tagtggagcc ttgcctatct tctttgactt 95 years old <210> 197 <211> 92 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Nov_DO2_ATGC_RNA <220> <221> misc_feature <222> (92)..(92) <223> RNA <400> 197 60. ggagaaagtt catgacactc tacgatttta ccagtggctg cgtctattta gtggagccat gcctatcttc tttgagtcgg tttgtctata au <210> 198 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Pcp4_120_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 198 tcagaaggca atgctcaggg ctgattcctt tgactcacat tgcgtctatt tagtggagcc atccctatct tctttgctag gtcccacaga acagc <210> 199 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pcp4_181_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 199 tccggcactt tgtctctcac ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 atccctatct tctttttgtc ttttccgttg gtcgc 95 <210> 200 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pcp4_305_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 200 actgagactg aatggccaca ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 atccctatct tcttttttct tctggaattt tctga 95 <210> 201 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pcp4_386_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 201 aacttggtgt cttcaggtgg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 atccctatct tctttttctt gatggatggt ggttg 95 <210> 202 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pcp4_472_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 202 ttcaggtttg tagcagggtg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 atccctatct tctttatggg tttctcttca tgcau 95 <210> 203 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_1081_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 203 ttctctgttg agtccgtcag tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttctata agaggaatga caatu 95 <210> 204 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_120_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 204 actttggtt ttcttcaggc tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttagcat ggacaatgct gcgaa 95 <210> 205 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_1216_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 205 tcacacacgg agccttttgt tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttagtct ggggcatagc tccca 95 <210> 206 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_273_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 206 60. catttaaggc aaatacatca tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc atcgctatct tctttgctat gctccccgct tgctu 95 <210> 207 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Pde1a_334_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 207 agatcatatc tggtaaagag tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc atcgctatct tctttgaatc ttgaagcggt tgata <210> 208 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Pde1a_469_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 208 atataatgca cagtttgagt tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tcttttgata cctgtatgaa gcatu 95 <210> 209 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_615_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 209 tgtacagaat agcaacgtcc tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttctcaa gcactgagcg gtcgu 95 <210> 210 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_759_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 210 ctgtcgctaa gaccatttca tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttctgaa aatgccctga catgu 95 <210> 211 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_904_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 211 ctgtagtgca acttccaagt tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttccatt agggccatgg tccau 95 <210> 212 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Pde1a_995_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 212 cttccgatca caaagtggag tctacgagtt tgcagtcacg tgcgtctatt tagtggagcc 60 atcgctatct tctttgactg ggcaaccatt gttga 95 <210> 213 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Penk_1282_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 213 caatactgag cttcaagact tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 gtacctatct tctttacaac atagccataa gagac 95 <210> 214 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Penk_286_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 214 catgggctgt aggagagaag tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 gtacctatct tctttcaaag cctcaggaac cgcgc 95 <210> 215 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Penk_638_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 215 gatatagctc gtccatcttc tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 gtacctatct tctttttctt cttctggctc catgg 95 <210> 216 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Penk_83_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 216 tgcctgggac tattctatct tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 gtacctatct tctttgtttc ctgctgttct agtga 95 <210> 217 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Penk_882_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 217 agttgggggc ttcttttgag tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 gtacctatct tctttgctct tttgcttcat cttcc 95 <210> 218 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Plcxd2_DO3_GAAA_RNA <220> <221> misc_feature <222> (89)..(89) <223> RNA <400> 218 gcactcctac acaatgactg attcctttga ctcacattgc gtctatttag tggagccgaa actatcttct tgaagatgg tgagggtau <210> 219 <211> 90 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Plcxd2_DO3_GGCC_RNA <220> <221> misc_feature <222> (90)..(90) <223> RNA <400> 219 60. gtggtagaaa atgagaacct gattcctttg actcacattg cgtctattta gtggagccgg ccctatcttc ttttgcttgt <210> 220 <211> 95 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> Rorb_2282_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 220 tcattcagaa ttggattcca ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aaatctatct tctttataac caccaaagtg August 95 <210> 221 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Rorb_4479_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 221 tcaatttct gccttaagcc ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aaatctatct tctttaagaa gaaaaagaag ttcau 95 <210> 222 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Rorb_536_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 222 atgtgaggtc atagataggt ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aaatctatct tctttggtaa acaagttggg tacag 95 <210> 223 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Rorb_6395_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 223 gagaaagtgt cacagatttg ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aaatctatct tctttaggta caattaagag aaagg 95 <210> 224 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Rorb_8435_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 224 tagttgttag ggagtgctgc ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 aaatctatct tctttaagta atagaaaact ctttu 95 <210> 225 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> Rorb_DO3_CCGG_RNA <220> <221> misc_feature <222> (98)..(98) <223> RNA <400> 225 agccttttaa agtcatattt ggtctgattc ctttgactca cattgcgtct atttagtgga 60 gccccgcta tcttcttaat cggtcatcat aaaatacu 98 <210> 226 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Rprm_654_RNA <220> <221> misc_feature <222> (87)..(87) <223> RNA <400> 226 cggtccgtga tggtgcgtc ttgtggtagc aaatatgcgt ctatttagtg gagccttgtc 60 tatcttcttt gacaggtttg cgttgcu 87 <210> 227 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Sst_432_AP1SWPLP_RNA <220> <221> misc_feature <222> (74)..(74) <223> RNA <400> 227 taacaggatg tgaatgtctt ctacgatttt accagtggcc tgactatctt cttttaggac 60 aacaatatta aagc 74 <210> 228 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Synpr_1071_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 228 catactagag actttaagct tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 attactatct tctttaaggt aatctatgca catta 95 <210> 229 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Synpr_1643_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 229 cctctctgga tgcaaagaaa tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 attactatct tctttaacta tggtgtctaa atctg 95 <210> 230 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Synpr_260_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 230 atgtacacga ccgtggcagc tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 attactatct tctttggtac ttgttctgga agaaa 95 <210> 231 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Synpr_591_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 231 aaggtatctc tgtccagagg tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 attactatct tctttgctt ctccatgggg tctga 95 <210> 232 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> Synpr_DO2_ATTA_RNA <220> <221> misc_feature <222> (98)..(98) <223> RNA <400> 232 cttaaaaatt cttctgctac tggtctacga ttttaccagt ggctgcgtct atttagtgga 60 gccattacta tcttcttcat tataattga ttgaaacu 98 <210> 233 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Yjefn3_138_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 233 cactagcgtg cccacatagt cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tttactatct tctttggcct tggtcacagc caccg 95 <210> 234 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Yjefn3_344_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 234 cttctcgcac tgcgtggtca cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tttactatct tctttgacag gaaggggatg tccau 95 <210> 235 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Yjefn3_686_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 235 aaactttcgg cggacgtcat cgtgcttgtg gtagcaaata tgcgtctatt tagtggagcc 60 tttactatct tcttttattt tggcaggtgc aggcc 95 <210> 236 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Bgn_412_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 236 gggcacagtc ttcagaccca tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 ctatctatct tctttgtgtc aggtgagatc tcctu 95 <210> 237 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Bgn_851_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 237 tcagggtctc agggagatct tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 ctatctatct tctttgtggt ccaggtgaag ttcgu 95 <210> 238 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Bgn_1194_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 238 tcccagtagg gcacagggtt tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 ctatctatct tctttggaag gtggcaggct gcacu 95 <210> 239 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Bgn_1577_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 239 aacaatggcg gtggcagtgt tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 ctatctatct tctttaggaa cacatgcctg gatgg 95 <210> 240 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Bgn_2309_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 240 tcagggaccc aggggtgagg tctacgattt taccagtggc tgcgtctatt tagtggagcc 60 ctatctatct tctttgacca tcacctccta ccaca 95 <210> 241 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Apq4_879_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 241 cttaaggcga cgtttgagct ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 cattctatct tctttgcggc tttgctgaag gcttc 95 <210> 242 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Apq4_2186_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 242 aattacactc acaatgccga ctgattcctt tgactcacat tgcgtctatt tagtggagcc 60 cattctatct tcttttaatt cacacaaatg ggtau 95 <210> 243 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Apq4_3100_RNA <220> <221> misc_feature <222> (95)..(95) <223> RNA <400> 243 cactggaaat gactgttaaa ctgattcctt tgactcacat tgcgtctatt tagtgga...

Claims

1. A method for detecting a target RNA sequence in a target RNA molecule in a sample, the method comprising: a) contacting the sample with a circularizable probe, which hybridizes to a target RNA sequence in the target RNA molecule; b) ligating the circularizable probe hybridized to the target RNA molecule using a ligase; wherein the target RNA molecule is used as a ligation template, the ligatable ends of the circularizable probe are juxtaposed to be ligated to each other, and the ligatable ends are ligated at the ligation site at or adjacent to the target RNA sequence, thereby circularizing the circularizable probe to produce a circularized probe, and wherein the circularizable probe comprises at least one ribonucleotide at the ligation site, and the circularized probe comprises DNA and comprises no more than 2 consecutive ribonucleotides; c) amplifying the circularized probe by rolling circle amplification (RCA) with a DNA polymerase to produce an RCA product, wherein the DNA polymerase is Bst polymerase or Phi29 DNA polymerase; as well as d) detecting the RCA product, thereby detecting the target RNA sequence.

2. The method of claim 1, wherein the circularizable probe comprises two or more parts, wherein circularizing the circularizable probe comprises ligation at at least two ligation sites. 3 . The method of claim 1 , wherein the circularizable probe comprises the at least one ribonucleotide at the ligatable 3′ end at the ligation site.

4. The method of claim 3, wherein the ligatable 3' end is created by extending the 3' end of the circularizable probe using the target RNA molecule as a template. 5 . The method of claim 1 , wherein the circularizable probe comprises the at least one ribonucleotide at the 3′ end of the circularizable probe. The method according to claim 1 , wherein the 3′ end and / or the 3′ penultimate nucleotide of the circularizable probe is a ribonucleotide.

7. The method of claim 1, wherein the circularizable probe does not contain a ribonucleotide at the 5' ligatable end of the circularizable probe.

8. The method of claim 1, wherein the 5' ligatable end of the circularizable probe is created by cleaving the circularizable probe hybridized to the target RNA molecule.

9. A method according to claim 8, wherein the first target-specific binding site of the circularizable probe is located inside the 5' end of the circularizable probe and the second target-specific binding site of the circularizable probe is located at the 3' end of the circularizable probe, so that after the circularizable probe hybridizes with the target RNA molecule, the additional sequence 5' of the first target-specific binding site forms a 5' flap, and the 5' flap is removed by cutting to create the 5' connectable end.

10. The method of claim 9, wherein one or more nucleotides at the 3' end of the additional sequence forming the 5' flap are complementary to homologous nucleotides in the target RNA molecule, wherein the nucleotides are capable of being replaced by the 3' ligatable end of the circularizable probe.

11. The method of claim 9, wherein the additional sequence comprises one or more ribonucleotides.

12. The method of claim 11, wherein the one or more nucleotides at the 3' end of the additional sequence are ribonucleotides.

13. The method according to any one of claims 9 to 12, wherein the 3'-most nucleotide of the additional sequence is a ribonucleotide.

14. The method of claim 1, wherein the circularizable probe is a single circularizable oligonucleotide comprising a first target-specific binding site at or within the 5' end of the circularizable probe and a second target-specific binding site at the 3' end of the circularizable probe, wherein the first target-specific binding site and the second target-specific binding site comprise or are processed to produce the 5' ligatable end and the 3' ligatable end of the circularizable probe, respectively.

15. A method according to claim 8, wherein the circularizable probe is a single circularizable oligonucleotide, which comprises a first target-specific binding site located inside the 5' end of the circularizable probe and a second target-specific binding site located at the 3' end of the circularizable probe, so that after the circularizable probe hybridizes with the target RNA molecule, the circularizable probe comprises an additional sequence 5' of the first target-specific binding site that does not hybridize with the target RNA molecule, wherein the nucleotides at the 3' end of the additional sequence are ribonucleotides, which are complementary to homologous nucleotides in the target RNA molecule and can be replaced by the 3' ligatable end of the circularizable probe, and wherein the additional sequence is removed by cutting to create the 5' ligatable end of the circularizable probe.

16. The method of claim 8, wherein the cleavage is performed by an enzyme having 5' nuclease activity or by a structure-specific nuclease.

17. The method of claim 16, wherein the enzyme is selected from the group consisting of Thermus aquaticus polymerase, Thermus thermophilus polymerase, and Thermus flavus polymerase.

18. The method of claim 8, wherein cleavage is performed by FLAP endonuclease.

19. The method of claim 1, wherein the ligase is T4 RNA ligase 1, T4 RNA ligase 2, or PBCV-1 DNA ligase.

20. The method of claim 1, wherein the method is used to detect variant bases in the target RNA molecule.

21. The method of claim 20, wherein the circularizable probe is a single circularizable oligonucleotide comprising a first target-specific binding site located within the 5' end of the circularizable probe and a second target-specific binding site located at the 3' end of the circularizable probe, such that after hybridization of the circularizable probe to the target RNA molecule, the circularizable probe comprises an additional sequence 5' to the first target-specific binding site that does not hybridize to the target RNA molecule, wherein the nucleotides at the 3' end of the circularizable probe are ribonucleotides, and wherein the nucleotides at the 3' end of the circularizable probe and the nucleotides at the 3' end of the additional sequence are identical to the same ribonucleotide. The invention relates to a method for preparing a 5' ligatable end of the circularizable probe and a nucleotide complementary to the nucleotides at the 3' end of the additional sequence, wherein when the nucleotides at the 3' end of the circularizable probe and the nucleotides at the 3' end of the additional sequence are complementary to the variant base, the nucleotides at the 3' end of the additional sequence are replaced by the 3' end of the circularizable probe and the additional sequence is removed by cutting, thereby generating a 5' ligatable end of the circularizable probe, and wherein when the nucleotides at the 3' end of the additional sequence and the nucleotides at the 3' end of the circularizable probe are not complementary to the variant base, the nucleotides at the 3' end of the additional sequence are removed by cutting, and the nucleotides at the 3' end of the circularizable probe do not hybridize with the target RNA molecule, thereby preventing connection.

22. The method of claim 20, wherein the circularizable probe is a single circularizable oligonucleotide comprising a first target-specific binding site located within the 5' end of the circularizable probe and a second target-specific binding site located at the 3' end of the circularizable probe, such that after hybridization of the circularizable probe to the target RNA molecule, the circularizable probe comprises an additional sequence 5' to the first target-specific binding site that does not hybridize to the target RNA molecule, wherein the nucleotide at the 3' end of the circularizable probe is a ribonucleotide, and wherein the nucleotide at the 3' end of the circularizable probe and the nucleotide at the 3' end of the additional sequence are complementary to the same nucleotide, wherein the 3' end of the circularizable probe is complementary to the nucleotide at the 3' end of the additional sequence. The nucleotide at the 5' end of the first target-specific binding site and the nucleotide at the 3' end of the additional sequence are complementary to the nucleotide at the 3' position of the variant base, and wherein the nucleotide at the 3' end of the additional sequence can be replaced by the 3' end of the circularizable probe, wherein when the nucleotide at the 5' end of the first target-specific binding site is complementary to the variant base, the additional sequence is removed by cutting, thereby producing a 5' ligatable end of the circularizable probe, and wherein when the nucleotide at the 5' end of the first target-specific binding site is not complementary to the variant base, the nucleotide and the additional sequence are removed by cutting, thereby producing a gap between the 5' ligatable end and the 3' ligatable end of the circularizable probe and preventing ligation.

23. A circularizable probe capable of hybridizing to a target RNA sequence in a target RNA molecule, wherein: (i) the probe comprises one or more parts, each part having at least one target-specific binding site, the at least one target-specific binding site being complementary to a cognate probe binding site at or adjacent to the target RNA sequence and hybridizing to the target RNA molecule, such that the ligatable ends of the circularizable probe or probe parts are juxtaposed for ligation to each other using the target RNA molecule as a ligation template, and the ligatable ends are ligatable at one or more ligation sites at or adjacent to the target RNA sequence, wherein ligation at the one or more ligation sites circularizes the circularizable probe; (ii) the circularizable probe comprises at least one ribonucleotide at the ligation site; and (iii) the circularisable probe comprises DNA after circularisation and comprises no more than 2 consecutive ribonucleotides, wherein the circularised probe is capable of being amplified by rolling circle amplification (RCA).

24. The circularizable probe of claim 23, wherein the circularizable probe is a single circularizable oligonucleotide comprising a first target-specific binding site located at or within the 5' end of the circularizable probe and a second target-specific binding site located at the 3' end of the circularizable probe, and wherein: (i) the second target-specific binding site comprises one or more ribonucleotides; and (ii) the first target-specific binding site is internal to the 5' end of the circularizable probe, and the circularizable probe comprises an additional sequence 5' of the first target-specific binding site, so that when the circularizable probe hybridizes with the target RNA molecule, the additional sequence forms a 5' flap, and the 5' flap can be cut to produce a ligatable 5' end, and the ligatable 5' end can be ligated to the 3' end of the circularizable probe to circularize the circularizable probe, wherein the additional sequence can optionally contain one or more ribonucleotides.

25. The circularizable probe of claim 23, wherein the probe comprises a backbone oligonucleotide comprising a first target-specific binding site located at or within its 5' end and a second target-specific binding site located at its 3' end; and one or more gap oligonucleotides, each of the one or more gap oligonucleotides comprising a target-specific binding site capable of hybridizing to the target RNA molecule between the first target-specific binding site and the second target-specific binding site of the backbone oligonucleotide, and wherein: (i) the second target-specific binding site of the backbone oligonucleotide and / or the target-specific binding site of at least one gap oligonucleotide comprises one or more ribonucleotides at the 3' end or within 5 nucleotides thereof; (ii) the first target-specific binding site is internal to the 5' end of the backbone oligonucleotide, the backbone oligonucleotide comprising an additional sequence 5' of the first target-specific binding site, such that when the backbone oligonucleotide hybridizes to the target RNA molecule, the additional sequence forms a 5' flap that can be cleaved to generate a ligatable 5' end that can be ligated to the 3' end of at least one gap oligonucleotide to circularize the probe, wherein the additional sequence can optionally contain one or more ribonucleotides; and (iii) one or more gap oligonucleotides optionally comprise an additional sequence 5' to the target-specific binding site, such that when the one or more gap oligonucleotides hybridize to the target RNA molecule, the additional sequence forms a 5' flap, which can be cleaved to produce a ligatable 5' end, which can be linked to the 3' end of another gap oligonucleotide or the 3' end of the backbone oligonucleotide, wherein the additional sequence can optionally contain one or more ribonucleotides.

26. A circularizable probe according to claim 24 or 25, wherein the circularizable probe or the backbone oligonucleotide and / or one or more gap oligonucleotides comprise an additional sequence 5' to the target binding site of the first target-specific binding site or the gap oligonucleotide, wherein the nucleotide at the 3' end of any additional sequence forming a 5' flap is complementary to a homologous nucleotide in the target RNA molecule that is identical to the nucleotide at the 3' end of the circularizable probe, or the 3' end of the backbone oligonucleotide and / or one or more gap oligonucleotides.

27. The circularizable probe according to claim 26, wherein the circularizable probe is used to detect variant bases in the target RNA molecule, and wherein: (i) the nucleotide at the 3' end of the circularizable probe or the backbone oligonucleotide and / or the one or more gap oligonucleotides and the nucleotide at the 3' end of the additional sequence are complementary to the variant base, and the nucleotide at the 3' end of the additional sequence can be replaced by the nucleotide at the 3' end of the circularizable probe or the backbone oligonucleotide or the one or more gap oligonucleotides, so that the additional sequence is removed by cleavage to produce a 5' ligatable end; or (ii) the nucleotide at the 3' end of the additional sequence and the nucleotide at the 3' end of the circularizable probe or the main chain oligonucleotide and / or the one or more gap oligonucleotides are not complementary to the variant base, so that the nucleotide at the 3' end of the circularizable probe or the main chain oligonucleotide and / or the one or more gap oligonucleotides cannot hybridize with the target RNA molecule, thereby preventing ligation, and the nucleotide at the 3' end of the additional sequence is removed by cleavage.

28. The circularizable probe according to claim 26, wherein the circularizable probe is used to detect variant bases in the target RNA molecule, and wherein: (i) the nucleotide at the 5' end of the first target-specific binding site or the 5' end of the target-specific binding site of the backbone oligonucleotide or one or more gap oligonucleotides is complementary to the variant base, the nucleotide at the 3' end of the circularizable probe or the backbone oligonucleotide and / or one or more gap oligonucleotides and the nucleotide at the 3' end of the additional sequence are complementary to the nucleotide at the 3' position of the variant base, and the nucleotide at the 3' end of the additional sequence can be replaced by the 3' end of the circularizable probe or the backbone oligonucleotide or one or more gap oligonucleotides, so that the additional sequence is removed by cleavage to generate a 5' ligatable end; or (ii) the nucleotide at the 5' end of the first target-specific binding site or at the 5' end of the target-specific binding site of the backbone oligonucleotide or one or more gap oligonucleotides is not complementary to the variant base, and the nucleotide and the additional sequence are removed by cleavage, thereby creating a gap between the 5' ligatable end and the 3' ligatable end of the circularizable probe and preventing ligation.

29. A set of two or more circularisable probes according to claim 27 or claim 28, wherein each circularisable probe in the set of circularisable probes comprises a different nucleotide at the 3' end of the circularisable probe and / or the backbone oligonucleotide or one or more gap oligonucleotides.

30. The set of circularizable probes according to claim 29, comprising three or four probes, wherein each circularizable probe comprises a different nucleotide at the 3' position of the variant base.

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