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 connectable probe comprising one or more moieties and hybridizing the probe with the target nucleic acid molecule, ligation of any probe that has hybridized with the target nucleic acid molecule, amplification of the ligated probe, and detection of the target nucleic acid molecule in the sample. 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 probe part comprises an additional sequence 5'of a target-specific binding site, the probe has an additional sequence 5'that does not hybridize to the target nucleic acid molecule after hybridization of the probe to the target nucleic acid molecule and forms a 5 'flap containing one or more nucleotides at its 3'end cleaved prior to ligation, and methods of synthesizing a DNA molecule with a Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide are provided. Probes for use in detection methods are provided.

Description

[0001] This application is a divisional application of the patent application with the application date of October 5, 2018, application number 2018800608475, and invention title "RNA-Templated Ligation". Technical Field

[0002] The present invention relates to the field of nucleic acid detection. Specifically, the present invention provides methods 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 ligatable chimeric DNA-RNA probe. Nucleic acid probes for such methods are also provided. Background Art

[0003] The detection of target nucleic acid sequences finds application in many different fields, including personalized medicine, cancer diagnosis and treatment, infectious diseases, and biosecurity.

[0004] Labeled hybridization probes complementary to the target nucleic acid sequence can be used to detect the target nucleic acid sequence, such as in Southern or Northern blot assays. Such probes allow for the 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 multiplex or automated detection assays.

[0005] The sensitivity of detecting target nucleic acid molecules can be enhanced by first amplifying the target sequence. Amplification techniques allow for the amplification of extremely low levels of the target sequence in a sample prior to detection, to increase the copy number of the target sequence in the sample. Amplification can be carried out using any of a variety of techniques available in the art, such as polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), and rolling circle amplification (RCA). However, these techniques are mainly directed to amplifying DNA sequences and typically require a DNA template for efficient performance.

[0006] PCR can be used to amplify target nucleic acid molecules and allows for exponential amplification using a pair of primers specific for sequences flanking the target nucleic acid sequence within the target nucleic acid molecule. However, target RNA sequences cannot be directly detected by PCR, and instead detection of RNA requires the formation of a cDNA intermediate using reverse transcriptase (RT-PCR), which can subsequently be amplified. Similarly, NASBA and LCR also require an initial reverse transcriptase step to generate a cDNA template for amplification (CA 2136764).

[0007] RCA utilizes strand displacement polymerase and requires a circular amplification template. Amplification of the circular template provides a cascade of RCA products that contain multiple copies of sequences complementary to the sequence of the amplification template, which can be ultimately detected. Circular RCA templates are typically provided by ligation reactions and can typically be provided by: using a target-specific probe as a cyclization template to circularize a target nucleic acid molecule, or by using a target nucleic acid molecule as a cyclization template to circularize a target-specific probe.

[0008] Circular RCA templates can be conveniently provided using symmetric selection probes, which contain longer nucleic acid strands suspended at both ends of a shorter nucleic acid strand, and the longer strands contain sequences complementary to sequences in the target nucleic acid molecule, as described in US 7883849. After hybridization, the selector and the target nucleic acid fragment are joined together by ligation to obtain a circular nucleic acid molecule. Alternative methods for selecting and amplifying nucleic acids involve general cyclization of genomic fragments, as described in Drmanac et al. 2010. Science 327, 78-81 and US 8518640. Selection probe-based methods for selecting target nucleic acid sequences are also provided in WO 03 / 044229. However, cyclization and amplification of RNA templates in this manner has not been demonstrated.

[0009] In alternative techniques, instead of circularizing the target nucleic acid, the target nucleic acid can be used as a template for ligating one or more probes. For example, so-called "padlock probes" can be used to detect target nucleic acid sequences, as described, for example, in US 5871921. Padlock probes are linear nucleic acid molecules that contain sequences complementary to the target nucleic acid molecule at their 3' and 5' ends, such that after hybridization of the padlock probe to its target nucleic acid molecule, the ends of the probe are juxtaposed for ligation. Adaptation of such methods has led to the development of techniques in which the ends of the probe bind to sequences separated by one or more bases in the target nucleic acid molecule, and the gap between the respective ends of the probe is filled using a nucleic acid polymerase (Hardenbol et al. 2003. Nat Biotechnol 21, 673-678), or using a linker (or gap) oligonucleotide (Weibrecht et al. Nature protocols 8, 355-372). Padlock probe-based detection techniques have recently been shown to be effective in detecting pathogens in samples.

[0010] However, ligation probe-based detection and amplification methods typically utilize DNA as the target and DNA probes, as this allows for a high level of sequence specificity and permits efficient amplification (and detection) of any ligation products formed. RNA templated ligation of DNA probes has been demonstrated and has been shown to be sensitive enough to distinguish SNPs within the RNA template (Nilsson et al. 2001. Nucleic acids research 29, 578-581). Other studies have also demonstrated the use of DNA ligation probes for the detection of specific miRNAs (Zhao et al. 2015. Acta Biochim Biophys Sin 47, 130-136; Jonstrup et al. 2006. RNA 12, 1747-1752). However, DNA ligation assays typically perform better using DNA templates rather than RNA templates (Nilsson et al. 2000. Nature Biotechnology 18, 791-793), and DNA ligation using RNA templates has been found to be inefficient and has also been found to have highly variable reaction kinetics even among closely related sequences. In addition, most ligases are intolerant to RNA templates, and ligases that have been shown to be tolerant have poor end joining fidelity, or in other words, a poor ability to distinguish probes that hybridize correctly to their target sequences. Thus, typically, methods in the art generate cDNA molecules from target RNA molecules prior to detection.

[0011] Target-dependent ligation of ribonucleotide probes or probes containing both ribonucleotides and deoxyribonucleotides has been shown to be useful for RNA targets (Zhao et al., supra). Specifically, the ligation efficiency of various combinations of heptameric ribonucleotide or deoxyribonucleotide probes using RNA ligation templates was tested, and ligation of such probes was shown to be templatable by RNA templates. However, amplification of ligation products containing both ribonucleotides and deoxyribonucleotides was not demonstrated. In addition, polymerases typically used in RCA methods have poor processivity on templates containing long stretches of ribonucleotides.

[0012] Accordingly, there remains a need for techniques that allow for the efficient detection of a broader range of nucleic acid targets (i.e., including RNA targets), which not only incorporate an efficient ligation step, but also permit amplification of the resulting ligation products in order to allow detection of the target. Although probes and methods have been developed primarily for the detection of RNA targets, certain novel probes and methods of the present invention have been found to also exhibit advantages in the detection of DNA targets, and thus they can be more widely applied. SUMMARY OF THE INVENTION

[0013] 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 consists mainly of DNA but contains ribonucleotides at specific positions relative to the ligation site, and these ribonucleotides are used to improve the efficiency and fidelity of ligation templated by the target nucleic acid molecule. Once the ligation product is formed, it is amplified, and the resulting amplification product is detected, thereby detecting the target RNA molecule.

[0014] Surprisingly, during the preparation of the present invention, it was observed that providing ribonucleotides rather than deoxyribonucleotides at or near the ligatable end of the probe enhances the ligation efficiency of the probe, and particularly results in an increase in ligation efficiency when using an RNA ligation template. This may also be beneficial in the context of detecting a DNA target when simultaneously detecting RNA and DNA targets (i.e., in the case of ligation of a probe templated by a DNA target). It is known that the ribose sugar-phosphate backbone in oligonucleotides prefers a specific conformation due to the presence of a 2'-OH that inhibits the C2'-endo conformation in ribose. When binding to a target RNA molecule, the specificity of the ligase for a probe containing ribonucleotides may also be higher than that for deoxyribonucleotides. Without wishing to be bound by theory, it is believed that the presence of ribonucleotides at or near the ligatable end of the probe allows one or more ends of the probe participating in templated ligation to be recognized and ligated by the ligase with higher efficiency and fidelity compared to the corresponding probe that does not contain ribonucleotides.

[0015] It was also observed that when the number of consecutive ribonucleotides in a ligation probe consisting mainly of DNA was kept to a minimum, the efficiency of amplifying the ligation product was not significantly inhibited. Thus, it was found possible to use a probe containing one or more ribonucleotides to improve the ligation efficiency while allowing for efficient amplification of the resulting ligation product.

[0016] Most broadly, 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:

[0017] (a) contacting the sample with a ligatable probe and hybridizing the probe with the target nucleic acid molecule;

[0018] (b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate any probe that has hybridized with the target nucleic acid molecule;

[0019] (c) amplifying the ligated probe from step (b) using a DNA polymerase; and

[0020] (d) detecting the amplification product from step (c), thereby detecting the target nucleic acid sequence;

[0021] Each probe is provided in one or more parts, each part having at least one target-specific binding site that is complementary to a homologous probe binding site at or adjacent to the target nucleic acid sequence and hybridizes to the target nucleic acid molecule such that, optionally after the step of cleaving the hybridized probe and / or extending the 3' end thereof using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe parts to ligate to each other, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and

[0022] wherein the probe contains at least one ribonucleotide at or near the ligation site, optionally after the cleavage and / or extension step, and the ligation probe consists mainly of DNA and contains no more than 4 consecutive ribonucleotides.

[0023] The detection method of the present invention can thus be used to detect a target DNA sequence in a target DNA molecule and / or a target RNA sequence in a target RNA molecule. Particularly preferably, the target nucleic acid sequence is an RNA sequence and the target nucleic acid molecule is an RNA molecule.

[0024] In one embodiment, the method includes using at least one probe directed against an RNA target sequence and at least one probe directed against a DNA target sequence to detect both RNA and DNA target sequences. Thus, target RNA and DNA molecules can be in the same sample. In this case, in the case of using an RNA ligase, it is beneficial for the DNA-targeted probe to also contain at least one ribonucleotide at or near the ligation site when hybridizing to the ligatable ends juxtaposed for ligation.

[0025] The present invention thus provides a method that allows for efficient ligation of probes containing both DNA and RNA nucleotides and subsequent direct amplification of the ligation product from the ligation reaction templated by the target nucleic acid molecule, i.e., without first providing a cDNA molecule complementary to the target molecule. This thus provides a detection method that is simplified relative to methods previously known in the art. In addition, in the context of detecting RNA, because the need to prepare a separate cDNA molecule is eliminated, detection of a target RNA sequence in a target RNA molecule can be more easily carried out in conjunction with detection of other nucleic acid sequences, such as a target DNA sequence. This can, for example, allow for simultaneous detection of target RNA and DNA sequences in the same sample.

[0026] A probe for such a method is also provided. More particularly, circularizable probes are of particular interest, and in this regard, the present invention thus provides a chimeric DNA-RNA padlock probe capable of binding and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein:

[0027] (i) The probe comprises one or more portions, each portion 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 with the target nucleic acid molecule, such that optionally after the step of cleaving the hybridized probe and / or extending its 3' end using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe portions to ligate to each other, creating one or more ligation sites at or adjacent to the target nucleic acid sequence, wherein ligation at the ligation site circularizes the probe;

[0028] (ii) The probe comprises at least one ribonucleotide at or near the ligation site; and

[0029] (iii) The probe consists mainly of DNA when ligated to form a ring and contains no more than 4 consecutive ribonucleotides.

[0030] Thus, in a particular 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:

[0031] a) contacting the sample with a padlock probe and hybridizing the probe with the target nucleic acid molecule;

[0032] b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate and thereby circularize any probe that has hybridized with the target nucleic acid molecule;

[0033] c) amplifying the ligated and circularized probe from step (b) by rolling circle amplification using a DNA polymerase; and

[0034] d) detecting the amplification product from step (c), thereby detecting the target nucleic acid sequence;

[0035] wherein each padlock probe is provided in one or more portions, each portion 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 with the target nucleic acid molecule, such that optionally after the step of cleaving the hybridized probe and / or extending its 3' end using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe portions to ligate to each other, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and

[0036] Wherein the probe comprises at least one ribonucleotide at or near the ligation site, optionally after the cleavage and / or extension step, and the ligated circularized probe consists mainly of DNA and contains no more than 4 consecutive ribonucleotides.

[0037] The probes of the present invention can be used to detect target DNA sequences in target DNA molecules and / or target RNA sequences in target RNA molecules, as discussed below. Thus, in a preferred embodiment, the probe is capable of binding to and detecting a target RNA sequence in a target RNA molecule.

[0038] Since the probe can be provided in one or more parts, there can be more than one ligation junction. In other words, one or more probe parts can each contain or generate (i.e., by cleavage or extension) ligatable 5' and 3' ends, and the probe as a whole can contain or generate one or more ligatable 5' ends and one or more ligatable 3' ends.

[0039] In certain embodiments, the probe can comprise two or more parts and can create two or more ligation junctions. The ligation junction can thus be provided between two different probe parts (or more particularly, between the ligatable ends of two different probe parts, or generated by the ends of two different probe parts), or provided between the two ends of a single-part probe or generated by the ends of a single-part probe.

[0040] After ligation, a probe is provided that comprises at least one ribonucleotide at or near the ligation site. The ligation site is the site where the terminal 5' phosphate group at the 5' ligatable end of the probe is joined to the terminal 3' hydroxyl group at the 3' ligatable end of the probe, and ligation can occur when the respective ligatable ends are directly juxtaposed and correctly hybridized to their respective complementary base pairs in the target nucleic acid molecule. Thus, once formed, the ligation site is the point of attachment between the nucleotides at the 3' and 5' ligatable ends of the probe or probe part, and its position is defined by the phosphodiester bond formed between these nucleotides. Thus, according to the methods and probes of the present invention, at least one nucleotide participating in the ligation reaction (i.e., the nucleotide providing the terminal 5' phosphate group or 3' hydroxyl group) or a nucleotide near the ligation site is a ribonucleotide. Preferably, at least one nucleotide at or near the ligatable 3' end at the ligation site is a ribonucleotide. In other words, in a preferred embodiment, when hybridized to a target nucleic acid molecule such that the 3' and 5' ends are juxtaposed for ligation, the probe comprises at least one ribonucleotide at or near the ligatable 3' end.

[0041] Thus it will be seen that upon ligation, the probe contains at least one ribonucleotide at or near the ligation site (or more particularly, at or near one or more ligation sites), the presence of which enhances the specificity and efficiency of ligation during the ligation reaction. According to the present invention, in the presence of two or more ligation junctions, at least one ribonucleotide may be present at or near one or more of these ligation junctions. It is not essential, but preferred, that at least one ribonucleotide is present at or near each ligation junction. The method of the present invention can thus be considered to provide a means of enhancing the ligation of a 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 ligation product containing no more than four consecutive ribonucleotides, the method also allows amplification of the ligation product without significant hindrance.

[0042] Any nucleotide at or near the ligation site can be a ribonucleotide rather than a deoxyribonucleotide. The term "near" in this context refers to a position within 5 nucleotides of the ligation site, i.e., 5 nucleotides 3' of the ligation site and 5 nucleotides 5' of the ligation site. Thus, any ribonucleotide within 5 nucleotides of the ligation site can be a ribonucleotide. The ribonucleotide can preferably be provided at a position within 4 nucleotides of the ligation site, or more preferably within 3 nucleotides of the ligation site. In yet another embodiment, the ribonucleotide can be within 2 nucleotides of the ligation site. Thus, the terminal nucleotide and / or the penultimate nucleotide at the 3' or 5' ligatable end can be a ribonucleotide. In certain embodiments, the penultimate nucleotide at the 3' or 5' ligatable end (preferably the 3' ligatable end) can be a ribonucleotide.

[0043] In a preferred embodiment, the ribonucleotide can be at the 3' or 5' ligatable end of the ligation site. Herein, the term

[0044] ‘at’ specifically refers to the terminal nucleotide at the end of the oligonucleotide, i.e., the nucleotide that provides the hydroxyl or phosphate group for ligation. Thus, in one embodiment, referring to the probe containing a ribonucleotide at the ligation site means that the terminal 3' nucleotide of the 3' ligatable end or the terminal 5' nucleotide of the 5' ligatable end is a ribonucleotide. Preferably, the 3' ligatable end at the ligation site is a ribonucleotide.

[0045] More than one nucleotide at or near the ligation site can be a ribonucleotide. Thus, in certain embodiments, more than one ribonucleotide at or near the ligation site (i.e., any one of 10 nucleotides within 5 nucleotides of the ligation site) can be a combined ribonucleotide, provided that the ligation probe contains no more than 4 consecutive ribonucleotides as discussed elsewhere.

[0046] In a preferred embodiment, the probe comprises at least one ribonucleotide at the ligatable end of the probe or a portion thereof. Preferably, the probe comprises at least one ribonucleotide at the 3' ligatable end at the ligation site. In other words, the nucleotide at the ligatable 3' end of the probe or a portion thereof is preferably a ribonucleotide rather than a deoxyribonucleotide.

[0047] In certain embodiments, the probe or probe portion does not comprise a ribonucleotide at or near the 5' ligatable end at the ligation site, and in particular does not comprise a ribonucleotide at the 5' ligatable end at the ligation site. In certain embodiments, where the 5' ligatable end is provided by the 5' end of the probe or probe portion (i.e., where the 5' ligatable end is not generated by cleavage of a hybridized 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 ligatable 5' end of the probe or a portion thereof is a deoxyribonucleotide. In such embodiments, the 3' hydroxyl group of the nucleotide (which is a ribonucleotide or a deoxyribonucleotide) at the ligatable 3' end from the probe or a portion thereof is linked to the 5' deoxyribonucleotide.

[0048] As outlined above, the above-described ligatable ends that ultimately form the ligation site need to be directly juxtaposed and hybridized to their respective complementary base pairs in the target nucleic acid molecule in order to effect ligation. However, depending on the design of the probe and / or a portion thereof, the ligatable ends can be juxtaposed in a variety of ways to effect ligation.

[0049] In a specific embodiment, the 3' end of the probe or a portion thereof is the ligatable 3' end at the ligation site. Thus, in this embodiment, the ligatable 3' end is provided at the 3' end of the probe (or alternatively expressed therefrom), 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.

[0050] Thus, in a preferred embodiment, the probe comprises at least one ribonucleotide at or near (but preferably at) the 3'-end of the probe or a portion thereof. Thus, the probe can not only comprise at least one ribonucleotide at or near the ligatable 3'-end at the ligation site, as described above, but the 3'-end of the probe or a portion thereof itself can be a ligatable 3'-end as described herein. Thus, in such an embodiment, the probe or probe portion provided in step (a) of the method of the invention can comprise at least one ribonucleotide at or near its 3'-end, which end hybridizes to the target nucleic acid sequence and is a ligatable 3'-end for the ligation reaction in step (b). In other words, in such embodiments, the nucleotide at the 3'-end of the probe or a portion thereof participates in the ligation reaction and is ligated to the ligatable 5'-end at the ligation site during the course of the detection method.

[0051] In another embodiment of the invention, the 5'-end of the probe is a ligatable 5'-end at the ligation site. Thus, the ligatable 5'-end can be provided at the 5'-end of the probe or a portion thereof, or in other words, the probe can hybridize to the target nucleic acid molecule such that the 5'-end of the probe or a portion thereof can be ligated to the ligatable 3'-end at the ligation site.

[0052] Thus, in a specific embodiment, two ligatable ends can be provided at the ends of the probe or probe portion, or in other words, the probe can hybridize to the target nucleic acid molecule such that, using the target nucleic acid molecule as a ligation template, the ligatable ends of the probe or probe portion are directly juxtaposed to ligate to each other without any further modification or processing (such as cleavage or extension) of the probe. In other words, the probe or each portion thereof can comprise at least one target-specific binding site at one or more of its ends such that each end of the probe (or a portion thereof) can hybridize to a directly adjacent homologous probe binding site in the target nucleic acid molecule such that ligatable ends are provided for ligation. In other words, in certain embodiments, step (b) can consist of a ligation step without any other steps to provide juxtaposed 5'- and 3'-ends of the probe or probe portion for ligation.

[0053] However, it is not necessary for the probe to comprise target-specific binding sites at its respective ends that hybridize to homologous sequences that are directly adjacent to each other in the target nucleic acid molecule in order to juxtapose the ligatable ends for ligation as described above, and optionally one or both of the ligatable ends of the probe can be generated only in one or more processing steps prior to ligation.

[0054] In a preferred embodiment of the invention, when the probe hybridizes to the target nucleic acid molecule (i.e., by cleavage of the hybridized probe), a 5'-ligatable end of the probe or a portion thereof can be created by cleavage.

[0055] Thus, the probe can comprise an additional sequence 5' of the probe or probe portion (or exterior) that provides a 5' ligatable end such that the 5' ligatable end of the probe or probe portion can only be created after the additional sequence has been cleaved (cut). In other words, the probe or probe portion can comprise a 5' additional sequence. Thus, the probe or probe portion can comprise a first target-specific target-binding site located internal to the 5' end of the probe or probe portion such that the probe or probe portion hybridizes to the target nucleic acid molecule and the portion that provides the ligatable 5' end at the ligation site (i.e., the target-specific binding site), when appropriate, comprises an additional sequence at its 5' end that does not hybridize to the target nucleic acid molecule. In this way, after any additional sequence 5' of the 5' ligatable end located at the 5' end of the probe or probe portion has been removed by cleavage, probe ligation can be carried out.

[0056] This additional sequence 5' of the first target-specific binding site that does not hybridize to the target nucleic acid molecule thus forms a 5' flap that can be removed by cleavage, thereby creating the 5' ligatable end of the probe or probe portion.

[0057] 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 the case of a two-part probe (such as a padlock probe comprising a backbone oligonucleotide and a gap oligonucleotide that hybridizes to a target between 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, both the 5' ends of the backbone and gap oligonucleotides can form 5' flaps. Additionally, a multi-part padlock probe can comprise more than one gap oligonucleotide. Thus, two or more gap oligonucleotides can each form a 5' flap.

[0058] Thus, after step (a), the method can comprise the step of cleaving any probe that has hybridized to the target nucleic acid molecule. This step removes any (or all) 5' flaps that are formed when the probe or probe portion hybridizes to the target nucleic acid molecule.

[0059] The step of cleaving the hybridized probe to remove the 5' flap can be carried out using an enzyme. Any enzyme capable of carrying out the reaction to remove 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 does not hybridize to the target nucleic acid molecule, but typically this will be an enzyme with 5' nuclease and / or structure-specific cleavage activity.

[0060] The 5' flap formed in this way can be recognized by structure-specific cleavage enzymes, such as enzymes that can recognize the junction between a single-stranded 5' overhang and a DNA duplex and cleave the single-stranded overhang. It should be understood that the branched triple-stranded structure that serves as the substrate for the structure-specific cleavage enzyme can be formed by the 5' end of one probe portion and the 3' end of another probe portion when both have hybridized to the target nucleic acid molecule, and by the 5' and 3' ends of one probe. Enzymes suitable for such cleavage include Flap endonucleases (FENS), which are a class of enzymes with endonuclease activity and capable of catalyzing the hydrolytic cleavage of phosphodiester bonds at single-stranded and double-stranded DNA junctions. Thus, in a preferred embodiment, the additional sequence 5' of the first target-specific binding site is cleaved by a structure-specific cleavage enzyme, such as Flap endonuclease. In a preferred embodiment, the enzyme can be a native or recombinant archaeal FEN1 enzyme from Pyrococcus furiosus (Pfu), Archaeoglobus fulgidus (Afu), Methanococcus jannaschii (Mja), or Methanothermobacter thermoautotrophicus (Mth), such as described in Ma et al. 2000. JBC 275, 24693-24700.

[0061] In other embodiments, enzymes that can recognize and degrade single-stranded oligonucleotides with free 5' ends can be used to cleave the additional sequence (5' flap) from the structure as described above. Thus, enzymes with 5' nuclease activity can be used to cleave the 5' additional sequence. Such 5' nuclease activity can be 5' exonuclease and / or 5' endonuclease activity. A 5' nuclease can recognize the free 5' end of a single-stranded oligonucleotide and degrade the single-stranded oligonucleotide. A 5' exonuclease degrades a single-stranded oligonucleotide with a free 5' end by degrading the oligonucleotide from its 5' end into constituent mononucleotides. 5' endonuclease activity can cleave the 5' flap sequence within one or more nucleotides. In addition, 5' nuclease activity can occur by the enzyme causing 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 (such as dinucleotides or trinucleotides), or cleaving the entire 5' single-stranded region, such as described for Taq DNA polymerase and its 5' nuclease in Lyamichev et al. 1999. PNAS 96, 6143-6148. Preferred enzymes with 5' nuclease activity include exonuclease VIII, or native or recombinant DNA polymerases from Thermus aquaticus (Taq), Thermus thermophilus, or Thermus flavus, or the nuclease domain therefrom.

[0062] In certain embodiments where the probe or probe portion contains 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 to the target nucleic acid molecule simultaneously with the 3'-ligatable end of the probe or probe portion (which may be a different probe portion as mentioned above). 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. Since they cannot hybridize to the target nucleic acid molecule and are prevented from doing so by the 3'-ligatable end of the probe or probe portion, the additional sequence can be regarded as forming a displacement flap, and the nucleotides within the additional sequence that are complementary to homologous nucleotides in the target nucleic acid molecule can be considered displacement nucleotides or displacement bases.

[0063] Thus, 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, where said nucleotides cannot hybridize to the target nucleic acid molecule simultaneously with the 3'-ligatable end of the probe or the 3'-ligatable end of another probe portion. In other words, 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'-ligatable end, or can be considered to be displaced from the target nucleic acid molecule. Thus, these nucleotides can be referred to as displacement nucleotides.

[0064] Before probe ligation can occur, any such additional sequence needs to be removed by cleavage. A probe having such an additional sequence at its (or one) 5'-end (i.e., a probe in which the first target-specific binding site is internal to the 5'-end of the probe or probe portion) can thus be considered to need to be activated (i.e., by cleavage) before ligation.

[0065] Surprisingly, for DNA and RNA target nucleic acid molecules, it has been found that the presence of ribonucleotides (i.e., the most 3'-nucleotide of the additional sequence) in the 5'-additional sequence of the probe, particularly at the 3'-end of the 5'-additional sequence, enhances the efficiency of removal (i.e., cleavage) of the additional sequence. This effect is specifically noted in the so-called invasive cleavage reaction described in more detail below. The additional sequence described above can thus contain 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 may be ribonucleotides. More particularly, one or more nucleotides at the 3'-end of the 5'-additional sequence that are complementary to homologous nucleotides in the target nucleic acid molecule (i.e., the displacement nucleotides as described above) may be ribonucleotides. In a specific embodiment, the additional sequence may consist entirely of ribonucleotides.

[0066] Enhanced cleavage of the 5' additional sequence of the probe is separate from the enhanced ligation efficiency, which is due to the presence of 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.

[0067] In another aspect, the present invention thus provides a method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising:

[0068] (a) contacting the sample with a ligation probe and hybridizing the probe to the target nucleic acid molecule;

[0069] (b) subjecting the sample to a ligation reaction using a DNA / RNA ligase to ligate any probe that has hybridized to the target nucleic acid molecule;

[0070] (c) amplifying the ligated probe from step (b) using a DNA polymerase; and

[0071] (d) detecting the amplification product from step (c) to thereby detect the target nucleic acid sequence;

[0072] 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 homologous probe binding site at or adjacent to the target nucleic acid sequence and hybridizes 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 internal to the 5' end of the probe or probe part such that after hybridization of the probe or probe part to the target nucleic acid molecule, the probe or probe part comprises an additional sequence 5' to the target-specific binding site that 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 the step of cleaving the hybridized probe to remove the 5' flap and optionally after the step of extending its 3' end using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe part to ligate to each other, thereby creating a ligation site at or adjacent to the target nucleic acid sequence.

[0073] In particular, the nucleotide at the 3' end of the 5' additional sequence can be a ribonucleotide. Optionally, one or more nucleotides near the 3' end of the 5' additional sequence can also be ribonucleotides.

[0074] 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, the method of this aspect of the present invention can also be used to detect more than one target sequence (including 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).

[0075] In another aspect, the present invention thus provides a probe for 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:

[0076] (i) the probe comprises one or more portions, each portion 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 the first target-specific binding site of the probe or the target-specific binding site of the probe portion is internal to the 5' end of the probe or probe portion, and the probe or probe portion comprises an additional sequence 5' to the target-specific binding site such that when the probe or probe portion 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 or probe portion, wherein the additional sequence contains one or more ribonucleotides (preferably at its 3' end) such that after the step of cleaving the hybridized probe or probe portion and optionally after the step of extending its 3' end using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe portion to ligate to each other, thereby creating one or more ligation sites at or adjacent to the target nucleic acid sequence.

[0077] In a preferred embodiment, the nucleotide at the 3' end of any additional sequence forming the 5' flap is complementary to the homologous nucleotide in the target nucleic acid molecule that is the same as 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 homologous nucleotides in the target nucleic acid molecule, wherein the nucleotides cannot hybridize to the target nucleic acid molecule simultaneously with the 3' ligatable end of the probe or the 3' ligatable end of another probe portion.

[0078] Such a probe may optionally be a single-piece or padlock probe of two (or more) pieces, or a probe comprising two or more pieces as defined elsewhere herein, except that it does not contain ribonucleotides at or near the ligation site.

[0079] Probes having 5' additional sequences that need to be removed by cleavage prior to ligation are well known in the art, for example in the form of invasive probes used in invasive assays. Invasive assays in the art typically involve using a first probe and a second probe having target-specific binding sites, wherein the first probe comprises a first target binding site internal to the 5' end of the probe. In such assays, the first probe and the second probe are typically designed to be complementary and capable of hybridizing to homologous binding sites in a target nucleic acid molecule such that the first probe hybridizes to the 5' portion of the target nucleic acid molecule and the second probe hybridizes directly juxtaposed to the first probe to the 3' portion of the target nucleic acid molecule and prevents the nucleotide at the 3' end of the additional sequence of the first probe from hybridizing to the target nucleic acid molecule. In other words, in an invasive assay, the nucleotide at the 3' end of the 5' additional sequence is the displaced nucleotide as described above. Since the second probe prevents the 3' end of the 5' additional sequence from hybridizing to the target nucleic acid molecule and the 5' additional sequence is subsequently cleaved, such methods are also referred to as invasive cleavage reactions.

[0080] Thus, the probes of the present invention can be invasive probes. Such probes can be particularly useful for detecting single nucleotide polymorphisms. The detection method of the present invention can therefore be used to detect single nucleotide polymorphisms or indeed any variant base in a target nucleic acid sequence. The probes used for such methods can be designed such that the 3' ligatable end of the probe is complementary to and capable of hybridizing to the nucleotide of interest (variant nucleotide) in the target molecule, and the nucleotide at the 3' end of the 5' additional sequence at the 5' end of the probe or at the 5' end of another different probe portion is complementary to the same said nucleotide but is prevented from hybridizing thereto by the 3' ligatable end (i.e., it is the displaced nucleotide as described above). Cleaving the probe to remove the additional sequence provides a 5' ligatable end that can be ligated to the 3' ligatable end of the probe or probe portion if the 3' ligatable end hybridizes correctly (i.e., is complementary) to the target nucleic acid molecule. Probes designed according to this principle provide a high degree of discrimination between different variants at the position of interest because only the probe whose 3' ligatable end is complementary to the nucleotide at the position of interest can participate in the ligation reaction. In one embodiment, the probe is provided as a single piece and the 3' and 5' ligatable ends are provided by the same probe.

[0081] Recently, alternative designs of invasive probes have been shown to provide results comparable to those of the design outlined above (Krzywkowski et al. 2017. Nucleic Acids Research 45, e161). Although this design is specifically disclosed in invasive assays utilizing padlock probes (the "invasive padlock" (iLock) probes described in detail below), certain features of such probes can generally be applied to invasive probes.

[0082] In this alternative design, the probe (which is provided as a probe with one part in the context of iLock probes) is designed such that the 3'-nucleotide of the 3'-target-specific binding site in the padlock probe (the second target-specific binding site as described herein) is complementary to and capable of hybridizing with the nucleotide in the 3' of the target nucleic acid molecule of the nucleotide of interest (i.e., the nucleotide immediately 3' of the variant nucleotide), and the 5'-nucleotide of the 5'-target-specific binding site in the padlock probe (the first target-specific binding site as described herein) is complementary to and capable of hybridizing with the nucleotide of interest in the target nucleic acid molecule (i.e., the variant nucleotide). Such a probe contains an additional sequence 5' to the first target-specific binding site such that the first target-specific binding site is internal to the 5'-end of the probe. The most 3'-terminal nucleotide of the additional sequence is complementary to the nucleotide in the 3' of the target nucleic acid molecule of the nucleotide of interest (i.e., immediately 3' of the nucleotide of interest / variant nucleotide), but is prevented from hybridizing with the target nucleic acid molecule by the 3'-ligatable end of the probe (i.e., the second target-specific binding site) and is thus a displacement nucleotide. As above, a similar probe design can be provided where the 5'-target-specific binding site and the 3'-target-specific binding site are provided by different probe parts.

[0083] Thus, more generally, a probe can be designed such that the 3'-ligatable end of the probe or probe part is complementary to and capable of hybridizing with the nucleotide in the 3' of the target molecule of the nucleotide of interest (variant nucleotide), and the nucleotide at the 3'-end of the 5'-additional sequence of the probe or at the 5'-end of another different probe part is complementary to the nucleotide 3' of the nucleotide of interest (the nucleotide immediately 3' of the variant nucleotide), but is prevented from hybridizing therewith by the 3'-ligatable end of the probe or probe part. Cleavage to remove the additional sequence provides a 5'-ligatable end that can be ligated to the 3'-ligatable end of the probe or probe part if the 5'-ligatable end hybridizes correctly (i.e., is complementary) to the target nucleic acid molecule. Probes designed according to this principle provide a high degree of discrimination between different variants at the position of interest, since only probes with a 5'-ligatable 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 as a single part, and the 3'- and 5'-ligatable ends are provided by the same probe.

[0084] Thus, in some embodiments, the methods of the present invention can be used to detect variant bases in a target nucleic acid molecule. In particular, probes according to any of the "invasion" designs mentioned above can be particularly useful for detecting variant bases in a target nucleic acid molecule.

[0085] In such invasion probes that create a 5' flap, in some embodiments it is preferred 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 most 3' nucleotide or at least the most 3' nucleotides of the additional sequence (or in other words, the 3' terminal nucleotide of the excised 5' flap) are ribonucleotides.

[0086] Invasion probes can be provided as padlock probes, which can consist of one or more parts, as further described below. Thus, a padlock probe can be a single-part padlock that hybridizes to a target nucleic acid molecule to form an "invasion" structure, or it can comprise two or more parts that hybridize to form one or more "invasion" structures with 5' flaps for cleavage.

[0087] In a specific representative embodiment, the probe for this method is a padlock probe provided as a single cyclizable oligonucleotide, which comprises a first target-specific binding site internal to the 5' end of the probe and a second target-specific binding site 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 nucleotide at the 3' end of the probe is a ribonucleotide and wherein the nucleotides at the 3' end of the probe and at the 3' end of the additional sequence are complementary to the same nucleotide, wherein when the nucleotides at the 3' end of the probe and at the 3' end of the additional sequence are complementary to a variant base, the nucleotide at the 3' end of the additional sequence cannot hybridize to the target nucleic acid molecule simultaneously with the 3' end of the probe and the additional sequence is removed by cleavage to produce a 5'-ligatable end of the probe, and wherein when the nucleotides at the 3' end of the additional sequence and at the 3' end of the probe are not complementary to the variant base, the nucleotide at the 3' end of the additional sequence is removed by cleavage and the nucleotide at the 3' end of the probe does not hybridize to the target nucleic acid molecule, thereby preventing ligation.

[0088] In another specific representative embodiment, the probe is a single circularizable oligonucleotide that includes a first target-specific binding site internal to the 5' end of the probe and a second target-specific binding site at the 3' end of the probe such that after hybridization of the probe to the target nucleic acid molecule, the probe includes an additional sequence 5' of the first target binding site that does not hybridize to the target nucleic acid molecule, wherein the nucleotide at the 3' end of the probe is a ribonucleotide, and wherein the nucleotides at the 3' end of the probe and at the 3' end of the additional sequence are complementary to the same nucleotide, wherein the nucleotide at the 3' end of the probe and the nucleotide at the 3' end of the additional sequence are complementary to the nucleotide 3' of the position of the variant base and wherein the nucleotide at the 3' end of the additional sequence cannot hybridize to the target nucleic acid molecule simultaneously with the 3' end of the probe, wherein when the nucleotide at the 5' end of the first target-specific binding site is complementary to the variant base, the additional sequence is removed by cleavage to generate a 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 cleavage to create a gap between the 5'-ligatable end and the 3'-ligatable end and prevent ligation.

[0089] In other embodiments, such invasive padlock probes can be provided similarly in two or more parts, such as padlock probes that include a backbone oligonucleotide and one or more gap oligonucleotides, as further described below.

[0090] In certain embodiments, the probe can be designed to bind two or more non-consecutive sequences within the target nucleic acid molecule. In other words, the probe or two probe portions can hybridize to a target nucleic acid molecule that has a gap between the corresponding target binding sites of the probe or probe portions. When the ends of the probe or probe portions have hybridized to leave a gap, the target nucleic acid molecule can be used as an extension template to fill this gap by extending the hybridized 3' end of the probe before ligating to the 5' end. After the 3' end of the probe has been extended to be adjacent to the 5'-ligatable end, the two ends can be ligated by a ligation reaction. In this way, a ligatable 3' end of the probe can be generated by extension.

[0091] Due to the incorporation of the complement of the target nucleic acid sequence into the ligation probe, this extension nick-fill implementation can be particularly useful for detecting highly variable target nucleic acid sequences. Thus, after amplification, detecting the amplified ligation probe (e.g., by sequencing) allows determination of the sequence of the target nucleic acid sequence. In other implementations, this nick-fill by extension can be combined with the feature of generating a ligatable 5' end by cleavage of the 5' flap. Thus, in the context of invasive probes, nick-fill extension can also be used to generate or provide a 3' ligatable end.

[0092] The 'extension nick-fill' implementation described above requires the incorporation of nucleotides into the extension product, and thus in such implementations, a mixture of nucleoside triphosphates is required in the reaction mixture for extension. In certain implementations, one or more of the provided nucleoside triphosphates (one, two, three, or all four nucleoside triphosphates) (nATP, nGTP, nCTP, dTTP / rUTP) can be ribonucleoside triphosphates. This can allow the incorporation of at least one ribonucleotide into the 3' ligatable end during the process of extending the 3' end of the probe. Thus, in one implementation, three of the provided nucleoside triphosphate molecules can be deoxyribonucleoside triphosphates, and the fourth nucleoside triphosphate molecule can be a ribonucleoside triphosphate. Preferably, based on knowledge of the target nucleic acid molecule sequence, one or more of the nucleoside triphosphates provided as ribonucleoside triphosphates can be selected such that the nucleotide at or near the 3' end of the resulting extension product after polymerization (e.g., the 3' terminal nucleotide of the 3' ligatable end at the ligation site) is a ribonucleotide. By way of representative example, if the nucleotide at the 5' end of the nick between two portions of the probe is known to be "G", a mixture of nucleoside triphosphates containing rCTP can be used, thereby providing an rCTP nucleotide at the 3' ligatable end of the extended probe.

[0093] In some implementations of extending the probe or a portion thereof to provide a 3' ligatable end, the probe (or portion) provided and hybridized to the target nucleic acid molecule can consist entirely of DNA, and ribonucleotides can be introduced by extension as described above. Thus, in such an implementation, a DNA probe can be provided and hybridized to the target molecule, and the ligatable probe hybridized to the target nucleic acid molecule can be converted into a chimeric DNA-RNA probe by an extension step. In other words, in the method of the present invention, the ligatable probe hybridized to the target molecule having juxtaposed ligatable 5' and 3' ends (i.e., ready to be ligated) can be a chimeric DNA-RNA probe.

[0094] In embodiments where ribonucleotides are introduced into a ligation probe via an extension reaction, ribonucleotides can be introduced at or near the ligation-capable 3'-end. For example, the terminus of the extension sequence and / or the penultimate 3'-nucleotide can be a ribonucleotide, and / or the extension reaction can introduce 5 or fewer nucleotides, or no more than 4 nucleotides.

[0095] Any convenient polymerase that can use RNA as an extension template, preferably a non-displacing polymerase, can be used to perform the 'gap-filling' extension described above, including RNA polymerases and DNA polymerases. Thus, an RNA-dependent RNA polymerase (RdRP), and / or an RNA-dependent DNA polymerase (RdDP) (e.g., reverse transcriptase) can be used in the 'gap-filling' embodiments described above.

[0096] In a preferred embodiment, the polymerase can be capable of incorporating both DNA and RNA residues into the extension product using an RNA template. For example, a mutant human mitochondrial DNA polymerase γ containing an E895A or E895G substitution has surprisingly been found to incorporate both rNTP and dNTP nucleoside triphosphates into the extension product (Kasiviswanathan et al. 2011. JBC 286, 31490-31500).

[0097] The probes used in the detection methods disclosed herein can be provided in one or more parts, each part containing at least one target-specific binding site that is complementary to a homologous probe-binding site at or adjacent to a target nucleic acid sequence in the target nucleic acid molecule. Thus, the probe (or each part thereof) is capable of hybridizing to the target nucleic acid molecule. The probe or probe part hybridizes to the target nucleic acid molecule in such a way that, using the target nucleic acid molecule as a ligation template, their termini are juxtaposed for ligation to each other, optionally following the step of cleaving the hybridized probe and / or extending its 3'-end using the target nucleic acid molecule as a template. Thus, the ligation reaction of step (b) of the detection method of the present invention comprises forming at least one phosphodiester bond between the respective ligatable 3'- and 5'-ends of adjacent probes or a part of a probe hybridized to the target nucleic acid molecule.

[0098] In certain embodiments, the probe can be provided in two or more parts, each such part having at least one target-specific binding site that is complementary to a homologous probe-binding site in the target nucleic acid molecule. Thus, in one embodiment, the probe can be provided in two parts, each part containing a target-binding site, and the ligation in step (b) comprises ligating the 3'-ligatable end of one part of the probe to the 5'-ligatable end of the other part. As described in more detail above, the respective ligatable ends of the probe can be generated by cleavage and / or extension prior to ligation.

[0099] In other embodiments of the present invention, the probe may be provided in two or more parts that create two or more ligation junctions, such as two, three, four, five, or more parts. Each part of the probe may provide (i.e., form or include) 3' and / or 5' ligatable ends that may be juxtaposed to ligate and are ligated in step (b) of the detection method of the present invention as outlined above. In certain embodiments, step (b) may thus include two or more separate ligation reactions, i.e., ligation reactions between adjacent ligatable ends provided by two or more parts of the probe.

[0100] 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 homologous probe-binding site in the target nucleic acid molecule. In such an embodiment, the ligation in step (b) includes the 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 the 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.

[0101] In a specific embodiment, one or more parts of the probe provided in more than two parts may be oligonucleotides for sequencing by ligation. In the context of the present invention, sequencing by ligation allows the detection of target nucleic acid molecules having an unknown sequence (e.g., a target nucleic acid sequence) flanked by known sequences at at least one end, thereby allowing determination of the unknown sequence. One or more parts of the probe as described herein may have target-specific binding sites that are complementary to homologous probe-specific binding sites in the target nucleic acid molecule and may further include nucleotides complementary to specific nucleotides in the unknown sequence at their 5' and / or 3' ends. The parts of the probe may ligate only when correctly hybridized to the nucleotides in the unknown sequence, and thus subsequent detection of the amplified ligation products may allow determination of the target nucleic acid sequence. In one specific embodiment, in the context of a multi-part padlock probe, one or more nicked oligonucleotides (discussed further below) may be oligonucleotides for sequencing by ligation.

[0102] In certain embodiments, and as briefly discussed above, the probes used in the detection methods of the present invention can be padlock probes that comprise one or more segments, which are optionally joined together after a cleavage step to form a circle. In a typical embodiment, a padlock probe can comprise a linear single-stranded oligonucleotide (backbone oligonucleotide) that comprises two target-specific binding sites that are complementary to homologous probe-binding sites in a target nucleic acid molecule, which are joined by a sequence (backbone or linker) that is not complementary to the target nucleic acid and thus provides 3' and 5' ligatable ends for a ligation reaction (optionally after cleavage and / or extension as discussed above). The 5' and 3' ends of the padlock probe can thus participate in one or more ligation reactions using the target nucleic acid molecule as a ligation template to form a circular nucleic acid molecule.

[0103] In certain embodiments, the padlock probe can be provided as a single cyclizable oligonucleotide that comprises a first target-specific binding site located at or internal to 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 respectively form or constitute the 5' ligatable end and the 3' ligatable end of the probe.

[0104] In such embodiments, the ligation can be direct, i.e., the 5' and 3' ends of the padlock probe can hybridize to target nucleic acid molecules that are directly adjacent to each other such that the 3' end of the padlock probe is ligated to the 5' ligatable end of the padlock probe to form a circular ligation product. Alternatively, in such embodiments, the 5' and 3' ends of the padlock probe can hybridize to target nucleic acid molecules with a gap between their target-specific binding sites, and the 3' end of the probe can be extended as described elsewhere herein to provide a 3' ligatable end of the probe for ligation to the 5' ligatable end of the padlock probe.

[0105] Padlock probes provided as a single cyclizable oligonucleotide may optionally include additional sequence 5' of its 5' target binding site, which may be cleaved to provide a 5' ligatable end at the ligation site. Thus, in one particular embodiment, the probe may be a single cyclizable oligonucleotide comprising a first target-specific target binding site internal to the 5' end of the probe and a second target-specific binding site at the 3' end of the probe such that after hybridization of the probe to the target nucleic acid molecule, the probe includes additional sequence 5' of the first target binding site that does not hybridize to the target nucleic acid molecule, wherein the nucleotide at the 3' end of the additional sequence is a ribonucleotide that is complementary to the homologous nucleotide in the target nucleic acid molecule but cannot hybridize to the target nucleic acid molecule simultaneously with the 3' ligatable end of the probe, and wherein the additional sequence is removed by cleavage to create the 5' ligatable end of the probe.

[0106] Padlock probes may alternatively be provided in two or more parts, i.e., as two or more oligonucleotides that are ligated to form a circular oligonucleotide. The first oligonucleotide may comprise a first target-specific binding site at or internal to its 5' end and a second target-specific binding site at its 3' end and may hybridize to a 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' ligatable end for ligation, a second oligonucleotide (and optionally third, fourth, etc. oligonucleotides) may be provided that comprises one or more target-specific binding sites complementary to homologous probe binding sites in the target nucleic acid molecule that are located between the respective homologous probe binding sites of the first target-specific binding site and the second target-specific binding site. In such an embodiment, cyclization of the padlock probe includes ligating the 5' and 3' ligatable ends of the first oligonucleotide to the 3' and 5' ligatable ends of these oligonucleotides (e.g., to the 3' and 5' ligatable ends of the second oligonucleotide, respectively). In such embodiments, the second oligonucleotide and subsequent oligonucleotides may be considered gap oligonucleotides. Padlock probes provided in two or more parts may thus be considered to comprise a backbone oligonucleotide (i.e., the oligonucleotide comprising the first target-specific binding site and the second target-specific binding site) and one or more gap oligonucleotides.

[0107] One or more gap oligonucleotides may be used for sequencing by ligation, as described above.

[0108] In some embodiments, the padlock probe (or more particularly, the backbone oligonucleotide of the padlock probe) itself may be provided in two or more parts, preferably two parts, which may be joined at a position different from the ligation site formed between the probe or probe parts that hybridize to the target nucleic acid molecule during cyclization of the padlock probe. In other words, the backbone oligonucleotide of the padlock probe may be provided in two or more parts, which may be joined at a position within a sequence that is not complementary to the target (i.e., the backbone or linker as described above). An example of a padlock probe with a backbone oligonucleotide provided in two parts is shown in Figure 17 In.

[0109] This ligation of the backbone oligonucleotide provided in two or more parts may be templated by a ligation template that is capable of hybridizing to each of the two or more parts of the backbone oligonucleotide, thereby juxtaposing the respective 5' and 3' ends for ligation. The ligation template thus contains regions complementary to the sequences at the ends of each of the two or more parts of the backbone oligonucleotide.

[0110] The ligation template may be a separate oligonucleotide, such as an oligonucleotide added to the sample together with or separately from the probe, or an oligonucleotide pre-hybridized to the probe, or it may be another target nucleic acid molecule, or another part of the same target nucleic acid molecule (i.e., another separate target sequence located at a different position within the same target nucleic acid molecule). Thus, the ligation template may be a synthetic or natural oligonucleotide and may be an RNA molecule or a DNA molecule.

[0111] The ligation of the parts of the backbone oligonucleotide may be direct or indirect, as defined elsewhere herein, or may require gap filling extension or other oligonucleotides (i.e., similar to gap oligonucleotides) that hybridize to the ligation template between the ends of the two parts of the backbone oligonucleotide. In addition, the ligation may require cleavage of a 5' appended sequence, optionally where the nucleotide at the 3' end of the appended sequence is a ribonucleotide, and / or where ribonucleotides are provided at or near the ligation site, as described elsewhere herein. It can be seen that this cyclization method (where the backbone oligonucleotide is provided in 2 or more parts) can be conveniently used to introduce ribonucleotides into the backbone oligonucleotide at a site different from the said (or one) ligation site on the (main) target molecule that serves as a template (e.g., by using rNTP extension). The presence of one or more such ribonucleotides at one or more different sites in the backbone oligonucleotide may be beneficial for the ligated (cyclized) padlock probe, as this serves to accelerate the RCA reaction, as discussed below.

[0112] In the case where more than one ligation occurs during step (b) (i.e., in the case where two or more ligation sites are formed), any ligation site can comprise at least one ribonucleotide as discussed in more detail above (i.e., the probe can comprise ribonucleotides at or near any ligation site). Thus, in the presence of two or more ligation sites, one or more of said ligation sites can comprise at least one ribonucleotide as defined herein. In a specific embodiment of the invention, the probe can comprise ribonucleotides at or near each ligation site. Thus, in a preferred aspect, any one or all of the portions of a probe provided in two or more portions can comprise at least one ribonucleotide at or near the ligatable 3' end at the ligation site, and in a specific embodiment, each portion of the probe can comprise at least one ribonucleotide at or near its ligatable 3' end at the ligation site. More preferably, each portion of the probe can comprise at least one ribonucleotide at or near its 3' end. However, it should be noted that in the case where the probe is provided in two or more portions, it is sufficient that only one of the two or more portions comprises at least one ribonucleotide at or near its ligatable 3' end or at its 3' end.

[0113] In the case where the probe is provided in two or more portions as described above, the 5' ligatable ends of any or all of the portions of the probe can be created by cleavage, as described elsewhere herein. In particular, the target-specific binding site of any or all of the portions of a probe provided in two or more portions can be located internal to the 5' end of the probe portion (i.e., can comprise additional sequence 5' of the target binding site that does not hybridize to the target nucleic acid molecule), and the additional sequence can be removed by cleavage prior to ligation. Similarly, any two or more portions of the probe can hybridize to non-contiguous sequences in the target nucleic acid molecule, and the gap therebetween is filled as needed by extending the 3' ends of any or all of the probes, as discussed above.

[0114] The number of ribonucleotides contained in the probe is not critical, as long as the probe meets the requirement that the ligation probe contains no more than 4 consecutive ribonucleotides. More particularly, the ligation probe can contain no more than 3 consecutive ribonucleotides, and in a preferred embodiment, no more than 2 consecutive ribonucleotides (e.g., 1 or 2 ribonucleotides). Since the probe can contain more than one ligation site, and ribonucleotides can be present at or near each or multiple ligation sites, it should be understood that ribonucleotides can be present at multiple (i.e., two or more) sites in the ligation probe, but at each site, there are no more than 4 (or 3 or 2) consecutive ribonucleotides. In other words, 1 ribonucleotide or no more than 4 (or 3 or 2) consecutive ribonucleotides can be present at each ligation site, and in fact, there is no restriction on the inclusion of ribonucleotides at other sites of the entire probe (i.e., not only at the ligation sites) within the scope described above. Surprisingly, it has been found that the presence of ribonucleotides in the circular ligation probe allows rolling circle amplification to proceed faster. The probe for use in the method of the present invention can thus optionally contain one or more (and at most 4 consecutive) ribonucleotides at one or more sites (including at one or more positions different from or near the ligation sites).

[0115] As described above, the probe or probe portion can 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) can contain one or more ribonucleotides that can be consecutive, and the number is not restricted; these ribonucleotides can be removed by cleavage such that when the ligation probe is formed, no more than 4 (or no more than 3 or 2) consecutive ribonucleotides remain in the probe.

[0116] In some embodiments where the probe is a padlock probe, the backbone oligonucleotide can consist entirely of DNA, and ribonucleotides can be contained in one or more gap oligonucleotides (more specifically, at or near their 3' ligatable ends), and / or they can be introduced by extension, as discussed above. In other embodiments, within the scope above, the backbone oligonucleotide can contain one or more ribonucleotides, for example, individual ribonucleotides can be scattered throughout. In another embodiment, the backbone oligonucleotide can contain at least one ribonucleotide at or near its 3' ligatable end. In such an embodiment, one or more gap oligonucleotides can also contain at least one ribonucleotide at or near their 3' ligatable ends. Alternatively, ribonucleotides can be provided only in the backbone oligonucleotide. Any combination of these features can be used.

[0117] The present invention can thus alternatively be seen as providing a method for detecting a target nucleic acid sequence in a target nucleic acid molecule that hybridizes to a ligatable chimeric DNA-RNA probe, the method comprising:

[0118] i) ligating a chimeric DNA-RNA probe that hybridizes to the target nucleic acid molecule;

[0119] ii) amplifying the ligated probe from step (i) with a DNA polymerase; and

[0120] iii) detecting the amplification product from step (ii), thereby detecting the target nucleic acid sequence;

[0121] 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 homologous 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 to each other for ligation 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

[0122] wherein the ligatable chimeric DNA-RNA probe comprises at least one ribonucleotide at or near the ligation site, and the ligated probe consists predominantly of DNA and contains no more than 4 consecutive ribonucleotides.

[0123] It is apparent that according to this method, for example after a cleavage and / or extension step, ligatable ends of probes or probe parts may have been generated that are juxtaposed for ligation, as described in more detail above.

[0124] The target nucleic acid molecule can be any sequence required for detection, analysis or amplification. Thus, it can be DNA or RNA or a modified variant thereof. Thus, the nucleic acid can consist of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotides capable of participating in Watson-Crick type or similar base pair interactions. Thus, the nucleic acid can be or can comprise, for example, bisulfite-converted DNA, LNA, PNA or any other derivative containing a non-nucleotide backbone.

[0125] The target nucleic acid molecule can thus be coding or non-coding DNA, such as genomic DNA or a sub-part thereof, or can be derived from genomic DNA, such as a copy or amplicon thereof, or it can be cDNA or a sub-part thereof, or an amplicon or copy thereof, etc.

[0126] As mentioned above, the target nucleic acid molecule can be a target RNA molecule. The target nucleic acid molecule can, for example, be an RNA molecule in an RNA pool or other nucleic acid molecule or nucleotide sequence, such as a human or from any source, from a transcriptome, or any other nucleic acid (such as organelle nucleic acid, i.e., mitochondrial or plastid nucleic acid), a naturally occurring or synthetic nucleic acid. The target RNA molecule can thus 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 typically can be an RNA molecule that is desired to be detected in a sample, in the sense that it is the target of an assay, i.e., the analyte. In a preferred embodiment, the target nucleic acid molecule is a microRNA (miRNA). In another preferred embodiment, the target RNA molecule is 16S RNA, preferably where the 16S RNA is from a microorganism (such as a pathogenic microorganism) in the sample and can be identified therefrom. Alternatively, the target RNA molecule can be the genomic RNA of a virus that has RNA as its genetic material, such as ssRNA or dsRNA. Notably, such viruses include Ebola virus, HIV, SARS, influenza, hepatitis C, West Nile fever, polio, and measles. Thus, the target RNA molecule can be sense RNA, antisense RNA, or double-stranded RNA from a viral genome, or sense RNA from a retroviral RNA genome.

[0127] The method of the present invention has particular advantages for detecting short target nucleic acid molecules, and particularly short target RNA molecules, which are generally difficult to amplify and characterize by conventional techniques known in the art. Thus, 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 the length is less than or at most 90, 80, 70, 60, 50, 40, 30, or 25 nucleotides. Particularly preferred such short nucleic acid molecules are miRNA molecules, which typically have a length of 19 - 25 nucleotides. A padlock probe having a pair of target-specific binding sites each containing 6 nucleotides is shown in WO 2015 / 071445, which can each bind to the target nucleic acid molecule and ligate in a target-dependent manner. Thus, shorter target (e.g., RNA) molecules, such as target molecules containing 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 within the range between any of the above integers.

[0128] The target nucleic acid molecule can be present in a sample. The sample can be any sample containing any amount of nucleic acid, from any source or of any origin, in which the target nucleic acid sequence in the target nucleic acid molecule needs to be detected. The sample can thus 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.

[0129] 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 formulations. The naturally occurring samples can be processed or treated before being subjected to the methods of the present invention. All biological and clinical samples are included, such as any cell or tissue sample of an organism, or any body fluid or formulation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples such as soil and water samples or food samples are also included. The samples can be freshly prepared, or they can be pretreated in any convenient manner, such as storage.

[0130] Representative samples thus include any material that can contain the target nucleic acid molecule, including for example food and related products, clinical and environmental samples. The sample can be a biological sample, which can contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material can thus include all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa, etc., or viruses. Representative samples thus include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, feces, cerebrospinal fluid or any other body fluid (e.g., respiratory secretions, saliva, milk, etc.), tissues, biopsies, cell cultures, cell suspensions, conditioned media or other samples of cell culture components, etc. The samples can be pretreated in any convenient or desired manner to prepare for the methods and uses of the present invention, such as by cell lysis or purification, isolation of nucleic acids or RNA, etc.

[0131] In a specific embodiment, the sample contains microbial cells or viruses that have been isolated 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 the microbial cells, for example a nucleotide sequence that can be characterized, differentiated, or identified at any level in the microbial cells or viruses, such as at the type, group, class, genus, species or strain level.

[0132] The probes described herein, and in fact one or more portions of the probes, may comprise one or more other sequences which may be used to introduce sequences into a ligation product, such as tags or detection sequences, such as barcodes or discriminator motifs, or binding sites for detection probes or primers. Such other sequences may be found, for example, at the 3' or 5' end of the probe or probe portion (preferably at the opposite end of the ligation-capable end of the probe or probe portion), or may be found in the middle of the probe or probe portion end, such as in a portion of a circularizable backbone oligonucleotide that does not hybridize to the target nucleic acid molecule. Tags such as barcodes or probe / primer binding sites may be designed for different requirements / purposes, for example, to introduce a common or general sequence so as to enable different ligation probes in a multiplex setting to be processed together, such as to introduce a binding site for a common or general amplification primer. This will enable different ligation probes to be amplified together, such as by PCR or RCA as library amplification. Alternatively or additionally, the tag / barcode sequences may be used to "label" different amplified ligation probes such that they can be easily distinguished from one another (i.e., "target" tags or markers), or to label different samples etc. such that they can be pooled prior to a common / general amplification step (i.e., "sample" tags or markers). Thus, in a multiplex setting, different tag sequences (such as different markers or detection sequences) may be provided for different probes (i.e., probes for different target nucleic acid molecules) and / or one or more common tag sequences may be provided for them, for example, for introducing a common or general sequence. Such methods may preferably be used in combination with sequencing by hybridization, sequencing by ligation or other next-generation sequencing chemistries, for example, for multiplex detection of multiple target nucleic acids in a sample.

[0133] The term "detection" is used herein broadly to include any means of determining the presence of a target nucleic acid sequence in a target nucleic acid molecule. It should be understood that in the methods of the present invention, the target nucleic acid is detected by detecting the amplified ligation probe (i.e., presence or absence) in a sample or any measured form of the amplified ligation probe. Thus, the amplification product of step (c) may be detected as a "reporter" of the target nucleic acid sequence. Thus, detecting the amplification product in step (d) may include determining, measuring, evaluating or assaying the presence or absence or quantity or location of the amplified ligation probe in any manner. The presence of the amplified ligation probe in the sample (i.e., confirmation of its presence or amount or ligation) indicates or identifies the presence of the target nucleic acid sequence, since the successful ligation of the probe (allowing amplification to occur) depends on the target nucleic acid molecule, and more particularly on the presence of the target nucleic acid molecule. Thus, detecting the amplified ligation probe allows determination of the presence of the target nucleic acid sequence.

[0134] Includes quantitative and qualitative determinations, measurements, or assessments, including semi - quantitative. Such determinations, measurements, or assessments can be relative (e.g., when detecting two or more different target nucleic acid molecules in a sample), or absolute. Thus, the term "quantitative" when used in the context of quantifying one or more target nucleic acid molecules in a sample can refer to absolute or relative quantification. Absolute quantification can be achieved by including one or more control RNA molecules of known concentration and / or by referencing the detection level of the target nucleic acid molecule to a known control RNA molecule (e.g., by generating a standard curve). Alternatively, relative quantification can be accomplished by comparing the detection levels or amounts between two or more different target nucleic acid molecules to provide relative quantification of each of the two or more different RNA molecules (i.e., relative to each other).

[0135] The sequence of the probe can be selected relative to the sequence of the probe and its target nucleic acid molecule. Thus, although the target - complementary region is selected relative to the target nucleic acid molecule to which it binds, the sequence of the remainder of the probe is not critical. However, the sequence should be chosen to avoid the occurrence of intramolecular hybridization. After the sequence is selected or identified, the probe can be synthesized using any convenient method.

[0136] As used herein, the terms "hybridization" or "hybridizing" refer to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex via Watson - Crick base pairing. Two nucleotide sequences are "complementary" when they share base - pair - wise homology. Thus, the complementary region in the probe region refers to the portion of that region that is capable of forming a duplex (or in other words, binding 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 variant of base pairing that also allows a third strand to wind around a double helix assembled in the Watson - Crick pattern to form a triple helix.

[0137] The amount of probe added to the sample can be selected to provide a sufficiently low concentration of the probe in the reaction mixture so as 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 significant or appreciable extent. However, generally, the probe will be used in an amount in excess of the target molecule. In representative embodiments, after combination 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, e.g., 1, 2, 5, 10, 20, 50 nM.

[0138] Multiple different probes can be added to a sample for multiplex assays (e.g., in the case where multiple different target nucleic acid molecules are present in the sample) in order to detect multiple target nucleic acid molecules in parallel. Multiplex assays may involve the detection of dozens, hundreds, thousands, or even tens of thousands of nucleic acid molecules in a sample. Thus, a multiplex assay can include at least 2 different probes, i.e., probes that are capable of hybridizing (directly or indirectly) to different first RCA products and thus detecting different analytes, for example. For example, a multiplex assay can utilize at least 2, 3, 4, 5, 10, 20, 30, 40, or 50 probes, such as 100, 200, 500, 1000, 10000, or more probes. In addition, the detection methods of the present invention (i.e., the detection methods for detecting target nucleic acid molecules) can be used in combination with broader methods for detecting other nucleic acid molecules in a sample. In a specific embodiment where the target nucleic acid molecule is a target RNA molecule, this can allow for the combined detection of the target RNA molecule with broader methods for detecting other (e.g., non-RNA) nucleic acid molecules in the sample.

[0139] After combining a sample containing a target nucleic acid molecule and one or more probes, the reaction mixture can be incubated for a period sufficient for 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 ligation probes (optionally after the steps of cleaving and / or extending the hybridization probes) are ligated to produce ligation products that can then be amplified, and the amplification products are detected to detect the target nucleic acid sequence.

[0140] In some representative embodiments, such as in in situ assays or other assays where the target nucleic acid molecules are immobilized, a washing step can be included between the addition of the probes and the ligation and / or amplification of the ligation products. In other words, the target nucleic acid molecules can be captured or immobilized on a solid support or substrate and washed to remove unbound or non-specifically bound probes. In some embodiments, a washing step can be included between the ligation of the probes and the amplification of the ligation products to remove unligated probes. In other representative embodiments, a washing step can be included before ligation.

[0141] In the case where the probe is a multi-part padlock, the backbone oligonucleotide can be contacted with the sample and hybridized, and then one or more nicking oligonucleotides (if used) can be added and hybridized. Alternatively, all parts of the multi-part probe can be added together.

[0142] Ligation involves the formation of a phosphodiester bond between the 3' OH group at the 3' end of a probe and the 5' phosphate group at the 5' end of the probe in two adjacent bases that hybridize to and are juxtaposed for ligation to a target nucleic acid sequence. Thus, depending on the design of the probe, the 3' OH group and / or the 5' phosphate group can be provided on ribonucleotides or deoxyribonucleotides. Accordingly, a ligase that can catalyze the formation of a phosphodiester bond in a target-specific manner can be selected.

[0143] The ligase can be a DNA ligase (i.e., a ligase characterized by its ability to catalyze 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 by its ability to catalyze 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 phosphodiester bonds between adjacent 3' ribonucleotides and 5' deoxyribonucleotides, and between adjacent 3' ribonucleotides and 5' ribonucleotides, in an efficient and target-dependent manner. Accordingly, both DNA and RNA ligases can be used in the methods of the present invention. Thus, reference to a DNA / RNA ligase includes DNA and RNA ligases that are capable of ligating hybridized 3' and 5' nucleotides to form a ligation product that contains ribonucleotides at or near the ligation site as described herein.

[0144] 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 RNA templated end joining fidelity of T4 DNA ligase and PBCV-1 ligase has only been characterized for DNA probes. Surprisingly, improved efficiency and fidelity of ligation were observed when the probes as defined herein were used for the detection of target nucleic acid sequences as compared to prior art DNA-only probes.

[0145] A suitable ligase and any necessary and / or desired reagents can be combined with the reaction mixture and maintained under conditions sufficient for ligation of the oligonucleotides to occur. Ligation reaction conditions are well known to those skilled in the art. During ligation, the reaction mixture can be maintained in some embodiments at a temperature in the range of about 4°C to about 105°C, about 4 to about 80°C, such as about 10 to about 70°C, about 15 to about 60°C, typically such as about 20°C to about 37°C for a period of time in the range of about 5 seconds to about 16 hours, such as about 1 minute to about 1 hour. In yet other embodiments, the reaction mixture can be maintained at a temperature in the range of about 35°C to about 45°C, such as about 37°C to about 42°C, for example at or about 38°C, 39°C, 40°C or 41°C for a period of time in the range of 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 a representative embodiment, the ligation reaction mixture comprises 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 of 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 employed.

[0146] Obviously, the ligation conditions can depend on the ligase used in the methods of the present invention. Thus, the ligation conditions described above are only representative examples and the parameters can be varied according to well-known protocols. However, it should be further understood that changing one parameter (e.g., temperature) may require modification of other conditions to ensure that other steps of the assay are not inhibited or disrupted, such as the binding of the probe to the target nucleic acid molecule. Such manipulation of the RCA assay method is routine in the art.

[0147] After the ligation probe is formed, 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 portion or portions of the ligation probe are amplified. The ligation probe (or portions thereof) can be amplified using any convenient method known in the art, such as PCR or variants thereof, SDA, HAD, LAMP or SMAP.

[0148] In a preferred embodiment, the circularization probe is connected. Preferably, in such embodiments, the probe can be amplified by rolling circle amplification (RCA). Rolling circle amplification using a circular template provides a tandem RCA product containing multiple tandem repeats, each repeat having a sequence complementary to the circular template oligonucleotide (i.e., the circularized ligation product). RCA can be carried out, for example, by contacting the sample with a separate primer that is complementary to and capable of hybridizing to the resulting circle, and the 3' end of the primer can be extended to provide a tandem RCA product. Alternatively, the target nucleic acid molecule can contain or provide the 3' end of a primer that can serve as a primer for RCA amplification.

[0149] The amplification of the ligation probe is carried out using a DNA polymerase, i.e., a polymerase capable of synthesizing DNA oligonucleotides from dNTPs. The term "DNA polymerase" as used herein includes any enzyme capable of incorporating dNTPs, and thus includes enzymes having DNA and RNA polymerase activities. In particular, the DNA polymerase can be an enzyme whose main activity or function is to synthesize DNA but is also capable of incorporating RNA nucleotides. A DNA polymerase capable of using a chimeric DNA-RNA oligonucleotide as an amplification template can be selected because the ligation probe contains at least one ribonucleotide. Representative polymerases for step (c) include Vent DNA polymerase and Bst DNA polymerase.

[0150] Surprisingly, it has also been found that Phi29 DNA polymerase is capable of synthesizing DNA oligonucleotides using a chimeric template oligonucleotide containing ribonucleotides, which includes incorporating deoxyribonucleotides into the synthesis product complementary to the ribonucleotides in the target nucleic acid molecule. In other words, Phi29 can act as a reverse transcriptase and incorporate DNA nucleotides (dNTPs) corresponding to the RNA bases complementary to the ribonucleotides in the template. The amplification of ligation probes containing up to four consecutive ribonucleotides using Phi29 DNA polymerase has been demonstrated. In the preferred embodiments of the various methods of the present invention defined above, the DNA polymerase is Phi29 DNA polymerase. However, this surprisingly new observed ability of Phi29 to accept RNA-containing substrates broadens the scope of Phi29-mediated amplification, and particularly RCA, and opens up new uses of enzymes other than the detection methods of the present invention described above. For example, Phi29-based RCA can be applied not only to nucleic acid detection but also to oligonucleotide synthesis and library preparation in next-generation sequencing technologies.

[0151] In one aspect, the present invention thus provides the use of Phi29 DNA polymerase as a reverse transcriptase.

[0152] Alternatively, this aspect 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 the DNA reverse complement thereof.

[0153] Although it has been reported that Phi29 DNA polymerase can be converted into an 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 way.

[0154] In particular, the present invention thus provides the use of Phi29 DNA polymerase as a reverse transcriptase, wherein the sequence of Phi29 DNA polymerase has not been modified to increase reverse transcriptase activity.

[0155] Furthermore, 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 the DNA reverse complement thereof, wherein the sequence of Phi29 DNA polymerase has not been modified to increase reverse transcriptase activity.

[0156] 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 Phi29 DNA polymerase and generating a DNA molecule comprising multiple tandem repeats of the reverse complementary sequence of the template nucleic acid molecule, wherein the sequence of Phi29 DNA polymerase has not been modified to enhance RT activity.

[0157] Referring to not modifying the sequence of Phi29 DNA polymerase 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 the enzyme acting on a template comprising one or more ribonucleotides and incorporating deoxynucleotides complementary to the ribonucleotides in the template into the synthesis product. Thus, the sequence has not been modified to confer or increase the reverse transcriptase activity of 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 no modifications have been introduced purposefully or intentionally to confer or increase the activity. More particularly, any sequence modifications that may exist relative to the wild-type or native Phi29 enzyme do not confer or increase RT activity.

[0158] By way of representative example, a structure-guided method for converting Phi29 DNA polymerase into a polymerase with RNA-dependent DNA polymerase activity is outlined in US2017 / 0159033, which proposes a number of amino acid substitutions to provide properties such as increased template polymerase stability or processivity, decreased exonuclease activity, and / or altered template specificity compared to the corresponding parental polymerase. Exemplary amino acids that can be substituted include those that may clash with an RNA / DNA heteroduplex, or those that may cause a steric clash between the polymerase and the 2'OH moiety of the template, and a number of exemplary substitutions are proposed. Other amino acid substitutions can be those that reduce or eliminate the exonuclease activity of Phi29 DNA polymerase, substitutions that increase the affinity for cognate bases in the active site, substitutions that inhibit primer strand binding to the exonuclease domain, substitutions that increase read length and / or processivity, substitutions that reduce the interpulse distance, and substitutions that enhance the thermal stability and / or stability of a binary complex comprising the polymerase and a nucleic acid substrate and / or a ternary complex comprising the polymerase, a nucleic acid substrate, and a cognate nucleotide or nucleotide analogue. Thus, preferably, the Phi29 DNA polymerase is not modified to affect any one or more of the foregoing properties, and it does not contain any one or more of the exemplary substitutions of the enzyme proposed in US2017 / 0159033 for converting Phi29 DNA polymerase into an enzyme with RNA-dependent DNA polymerase activity.

[0159] However, the Phi29 DNA polymerase according to this aspect of the invention may, according to certain embodiments, contain one or more alternative modifications introduced for different purposes (i.e., other than increasing its reverse transcriptase activity), such as to alter one or more of its other biochemical properties, and / or to enhance the recombinant expression and / or purification of the enzyme (i.e., to increase its yield).

[0160] According to certain embodiments, the sequence of the Phi29 DNA polymerase may not be modified relative to the wild-type sequence. According to one specific embodiment, the sequence of the Phi29 DNA polymerase may thus be a wild-type or native sequence. In other words, the sequence of the Phi29 DNA polymerase may be a non-mutated sequence.

[0161] In one 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.

[0162] The template nucleic acid molecule thus contains at least one ribonucleotide, and the method according to this aspect of the invention includes generating a DNA molecule that contains or has a sequence complementary to the template nucleic acid molecule.

[0163] In these respects, it is preferred that the template nucleic acid molecule contains no more than 4, and preferably no more than 3 or 2 consecutive ribonucleotides (see the discussion above, which also applies to this context). Although in some cases better results may be obtained when the number of consecutive ribonucleotides does not exceed 2, it should be understood that conventional optimization of the method is possible, especially in the case of circular templates, such that it is not necessary to limit the number of consecutive ribonucleotides to no more than 2. In certain embodiments, the template nucleic acid molecule can thus contain 1, 2, 3, or 4 consecutive ribonucleotides.

[0164] This is demonstrated in the examples below. The template molecule can contain ribonucleotides at multiple sites, and the total number of ribonucleotides is not critical or limiting. Thus, the template nucleic acid molecule can contain no more than 4 (i.e., 4, 3, 2, or 1) ribonucleotides at each of two or more separate or distinct sites. In one embodiment, the template nucleic acid molecule can contain one or more single ribonucleotides, i.e., ribonucleotides flanked by deoxyribonucleotides on their 5' and 3' sides, at more than one position as described elsewhere herein. Each ribonucleotide site is thus separated by one or more deoxyribonucleotides in between. In certain embodiments, two sites can be separated by only a single deoxyribonucleotide.

[0165] In yet another embodiment, the template nucleic acid molecule can contain ribonucleotides at only a single site within the template nucleic acid molecule. In a specific embodiment, the template nucleic acid molecule can contain only a single ribonucleotide.

[0166] The consecutive ribonucleotides can 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 (i.e., C and U) in the amplification template.

[0167] In this particular aspect of the invention, it is preferred that the template nucleic acid molecule contains at least one ribonucleotide, rather than its chemical modifications, analogs, or derivatives. 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).

[0168] The factors necessary for optimal reverse transcriptase activity of Taq and Bst polymerases described above, such as salts or specific metal cations such as Mn2 + + concentration, can be adjusted to optimize Phi29 reverse transcriptase activity. The activity of Phi29 towards longer stretches of ribonucleotides and / or purine RNA bases can be enhanced by altering the RCA reaction conditions.

[0169] Of course, chimeric template molecules containing RNA and DNA bases can be generated by ligating the probes according to the invention. However, this aspect of the invention is not limited to using Phi29 for amplifying the ligated probes according to the methods herein, and includes any chimeric molecule containing ribonucleotides and deoxyribonucleotides for amplification. Such chimeric DNA-RNA molecules can be circular molecules and can be any circular or circularized probe. Thus, in any case, Phi29 polymerase can be used to amplify any circularized probe generated anyway (such as padlock probes, iLock, molecular inversion probes, selection probes, etc.). For example, the probe can be a molecular inversion probe with an RNA-filled gap, or a selection probe hybridized to a target molecule containing RNA, etc. Thus, in certain preferred embodiments, the template nucleic acid molecule can be a circular template, for example, formed according to any method disclosed herein.

[0170] Other fields of application can include oligonucleotide production and next-generation sequencing as mentioned above, as well as RNA-based therapies.

[0171] Generally, any convenient protocol capable of detecting the presence of the amplified ligation product can be used in the detection method of the present invention. The detection protocol may or may not require a separation step. Referring to the detection of the amplified ligation product herein means detecting the sequence (i.e., its complementary sequence) formed by the ligation of the probe and / or its complement.

[0172] The amplified ligation product (i.e., the amplification product) can be detected using any convenient protocol, where the specific protocol employed 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 hybridizing a labeled detection oligonucleotide that hybridizes to the amplified ligation product. Alternatively, the amplification product can be detected indirectly, such as by PCR amplifying the product and detecting the amplification product.

[0173] Representative non-specific detection schemes of interest include those employing a signal generation system that selectively detects single- or double-stranded DNA products, for example, via intercalation. Representative detectable molecules that can be used in such embodiments include fluorescent nucleic acid stains (such as phenanthridinium dyes), including their monomers or homodimers or heterodimers, which produce enhanced fluorescence when complexed with nucleic acids. Examples of phenanthridinium dyes include ethidium homodimer, ethidium bromide, propidium iodide, and other alkyl-substituted phenanthridinium dyes. In another embodiment of the present invention, the nucleic acid stain is or incorporates an acridine dye or its homodimer or heterodimer, such as acridine orange, acridine homodimer, ethidium, acridine heterodimer, or 9-amino-6-chloro-2-methoxyacridine. In yet another embodiment of the present invention, the nucleic acid stain is an indole or imidazole dye, such as Hoechst 33258, Hoechst 33342, Hoechst 34580 (BIOPROBES 34, Molecular Probes, Inc., Eugene, Oregon (May 2000)), DAPI (4',6-diamidino-2-phenylindole), or DIPI (4',6-(diimidazolin-2-yl)-2-phenylindole). Other permitted 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 (for example, incorporating Tb 3+ and Eu 3+)。In certain embodiments of the present 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 acid. Any of the dyes described in U.S. Patent No. 4,883,867 (1989) to Lee, U.S. Patent No. 5,582,977 (1996) to Yue et al., U.S. Patent No. 5,321,130 (1994) to Yue et al., and U.S. Patent No. 5,410,030 (1995) to Yue et al. (all four patents are incorporated by reference), including nucleic acid stains that are commercially available from Molecular Probes, Inc., Eugene, Oregon, under the trademarks TOTO, BOBO, POPO, YOYO, TO-PRO, BO-PRO, PO-PRO, and YO-PRO, can be used. Any of the dyes described in U.S. Patent No. 5,436,134 (1995) to Haugland et al., U.S. Patent No. 5,658,751 (1997) to Yue et al., and U.S. Patent No. 5,863,753 (1999) to Haugland et al. (all three patents are incorporated by reference), including nucleic acid stains that are commercially available from Molecular Probes, Inc., Eugene, Oregon, under the trademarks SYBR Green, EvaGreen, SYTO, SYTOX, PICOGREEN, OLIGREEN, and RIBOGREEN, can be used.

[0174] In still other embodiments of the present invention, the nucleic acid stain is a monomeric, homodimeric, or heterodimeric cyanine dye that incorporates an azo or polyazo benzindolium heterocycle, such as azobenzoxazole, azobenzimidazole, or azobenzothiazole, which provides enhanced fluorescence when associated with nucleic acid, including nucleic acid stains that are commercially available from Molecular Probes, Inc., Eugene, Oregon, under the trademarks SYTO, SYTOX, JOJO, JO-PRO, LOLO, and LO-PRO.

[0175] In still other embodiments, a signal generation system that is typically specific for the amplified ligation product rather than the nucleic acid molecule can be used to detect amplification. In these embodiments, the signal generation system can include a nucleic acid or oligonucleotide that specifically binds to a sequence found in the amplified ligation product (i.e., the reporter domain sequence), wherein the nucleic acid / oligonucleotide can be labeled with a label that can be detected directly or indirectly. In a specific embodiment, the amplified ligation probe can be detected by ligation sequencing.

[0176] A directly detectable label is a label that can be detected directly without the use of other reagents, while an indirectly detectable label is a label that can be detected by using one or more additional reagents, such as a member of a signal generating system in which the label consists of two or more components.

[0177] In many embodiments, the label is a directly detectable label, and directly detectable labels of interest include, but are not limited to: fluorescent labels, radioisotope labels, chemiluminescent labels, etc. In many embodiments, the label is a fluorescent label, and the labeling reagent used in such embodiments is one or more fluorescently labeled nucleotides, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP), etc. Fluorescent moieties that can be used to label nucleotides to produce a labeled probe nucleic acid (i.e., a detection probe) include, but are not limited to: fluorescein, cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy630 / 650, etc. As is known in the art, other labels can also be employed, such as those described above.

[0178] 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 of altering the fluorescence emission of a fluorophore 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. Patent No. 6,248,526, the disclosure of which is incorporated herein by reference (and 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. Patent 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. Patent 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. Patent No. 5,989,823, the disclosure of which is incorporated herein by reference) and conformation-assisted probes (as described in provisional application serial number 60 / 138,376, the disclosure of which is incorporated herein by reference).

[0179] 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.

[0180] 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.

[0181] 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.

[0182] The next step of the method is to detect a signal from the labeled product of interest, where the signal detection may vary depending on the particular signal generation system employed. In some embodiments, only a detectable signal (e.g., fluorescence) is determined and used in the subject assay, such as to determine or identify the amplified ligation product and thus the presence or absence of the target nucleic acid molecule. Depending on the particular label employed, the signal detection can indicate the presence or absence of the target nucleic acid sequence in the target nucleic acid molecule.

[0183] In those embodiments where the signal generation system is a fluorescence signal generation system, signal detection typically includes detecting a change in the fluorescence signal from the reaction mixture to obtain an assay result. In other words, any modulation of the fluorescence signal generated by the reaction mixture is evaluated. Depending on the nature of the label employed, the change can be an increase or decrease in fluorescence, but in some embodiments is an increase in fluorescence. Any convenient means can be used, such as a suitable fluorometer, such as a thermostable cuvette or a plate reader fluorometer, to screen for fluorescence enhancement of the sample, or for example, when the sample is a tissue sample on a microscope slide, the fluorescence can be detected using a fluorescence microscope. Fluorescence is suitably monitored using a known fluorometer. Signals from these devices (e.g., in the form of photomultiplier tube voltage) are sent to a data processor board and converted into a spectrum associated with each sample tube. Multiple tubes, such as 96 tubes, can be evaluated simultaneously. Thus, in some embodiments, multiple analytes can be detected in parallel, while in other embodiments, multiple analytes can be detected sequentially, such as one analyte at a time or one group of analytes at a time.

[0184] In the case where the detection protocol is a real-time protocol, such as that employed in a real-time PCR reaction protocol, data can be collected in this manner at frequent intervals throughout the reaction, such as every 3 minutes. By monitoring the fluorescence of the reactive molecules from the sample during each cycle, the progress of the amplification reaction can be monitored in various ways. For example, the data provided by the melting peak can be analyzed, such as by calculating the area under the melting peak, and these data can be plotted relative to the cycle number.

[0185] The spectra generated in this manner can be resolved, for example, using a "fit" of a preselected fluorescence fraction (such as a dye) to form peaks representative of each signal generating moiety (i.e., fluorophore). The area under the peak can be determined, which represents the intensity value of each signal, and if desired, can be expressed as a quotient of one another. Differences in signal intensity and / or ratio will allow the recording of changes in the labeled probe throughout the reaction or under different reaction conditions (e.g., temperature). This change is related to the binding phenomenon between the oligonucleotide probe and the target sequence or the degradation of the oligonucleotide probe that has bound to the target sequence. Integration of the differential area under the peak will allow the calculation of the intensity value of the labeling effect.

[0186] Fluorescence changes in the screening mixture provide one or more assay results, depending on whether the sample is screened once at the end of the primer extension reaction or multiple times, for example, after each cycle of the amplification reaction (e.g., as performed in real-time PCR monitoring). The data generated as described above can be interpreted in various ways. In its simplest form, during or at the end of the amplification reaction, an increase or decrease in fluorescence from the sample indicates an increase in the amount of the target analyte present in the sample, e.g., as associated with the amount of amplified ligation product detected in the reaction mixture, indicating that the amplification reaction has occurred and thus the fact that the target nucleic acid molecule was actually present in the initial sample. Quantification can also be performed by monitoring the amplification reaction throughout the amplification process. Quantification can also include assaying one or more nucleic acid controls in the reaction mixture, as described above.

[0187] In this way, the presence of the amplification product in the reaction mixture can be readily screened (or evaluated or assayed, etc.) and thus the presence of one or more target nucleic acid molecules can be screened. The method is applicable to detecting a single target nucleic acid molecule as well as multiple nucleic acid molecules, where two or more different target nucleic acid molecules are assayed in the sample. In these latter multiplex cases, the number of different probes that can be employed typically ranges from about 2 to about 20 or more, e.g., up to 100 or more, 1000 or more, etc., where multiple analytes in the sample can be detected in parallel or sequentially. Analyzing multiple analytes simultaneously and in a single reaction using multiple different probes (multiplexing) can be enhanced by increased sensitivity and, in some embodiments, increased specificity, which can be obtained using the methods and probes of the present invention. Each probe set can be designed to produce a ligation product that can be used to determine the presence or absence, quantity, and / or location of the analyte ultimately probed by the probe.

[0188] Any well-established method known from the literature for analyzing nucleic acid molecules can be used to detect the amplified ligation probe, including liquid chromatography, electrophoresis, mass spectrometry, microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric assays (such as ELISA), flow cytometry, mass spectrometry (CyTOF), etc.

[0189] Detecting the amplified ligation probe further includes determining the sequence of the amplified ligation probe. Thus, in certain embodiments, detection can include sequencing all or part of the amplified ligation probe. The probes and methods of the present invention can be used homogeneously (i.e., in solution) as described above, or alternatively, used heterogeneously on a solid phase, e.g., where the target nucleic acid molecule becomes immobilized on a solid phase, thus allowing the use of wash steps. This can be due to, for example, immobilization 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: The wash steps can help remove unbound and / or unligated probes, etc., inhibitory components, and can enrich the target molecules from an undesired large-volume sample. Higher concentrations and amounts of probes can be used because unbound probes and RNA molecules can be removed by washing.

[0190] In a preferred embodiment of the present invention, the target nucleic acid molecule is detected in situ. This can allow direct detection of the level, location, or localization of the target nucleic acid molecule in the sample. The sample can thus preferably be any sample that reflects the normal or native ("in situ") localization of the target nucleic acid molecule, i.e., any sample in which the target nucleic acid molecule is normally or naturally present. Such a sample will advantageously be a cell or tissue sample. Particularly preferred are samples such as: cultured or harvested or biopsied cell or tissue samples, where the target nucleic acid molecule can be detected to reveal the localization 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, e.g., on a microscope slide. The samples can be freshly prepared, or they can be pretreated in any convenient manner, such as by fixation or freezing. Thus, fresh, frozen, or fixed cells or tissues can be used, e.g., FFPE tissue (formalin-fixed paraffin-embedded).

[0191] In alternative embodiments, the target nucleic acid molecule can be immobilized. Immobilizing the target nucleic acid molecule on a solid phase can be achieved in a variety of ways. Thus, 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 an amplified ligation product can be generated such that it is ligated to the target nucleic acid molecule, e.g., by means of a primer for amplification that is the target nucleic acid molecule or is ligated to the target nucleic acid molecule, as described elsewhere herein. Alternatively, the amplified ligation product can simply be immobilized to a solid support. For example, prior to amplification, the primer for amplification can have a fixable group or moiety or fixation member, or can be immobilized.

[0192] The immobilized capture probe, target nucleic acid molecule, primer for amplification, or amplified ligation product can be immobilized in any convenient manner, i.e., bound to a support. Thus, the immobilization method and means, as well as the solid support, can be selected according to choice from any number of immobilization components and solid supports widely known in the art and described in the literature. Thus, the capture probe, target nucleic acid molecule, primer for amplification, or amplified ligation product can be directly bound to the support (e.g., chemical cross-linking), it can be bound via a linking group, or via one or more intermediate binding groups (e.g., via biotin-streptavidin interaction). Thus, the capture probe, target nucleic acid molecule, primer for amplification, or amplified ligation product can have a component for immobilization provided thereon (e.g., an affinity binding partner, such as biotin or hapten or nucleic acid molecule, capable of binding its binding partner, i.e., a cognate binding partner, such as streptavidin or antibody or nucleic acid molecule). The capture probe can be immobilized before or after binding the analyte. In addition, such "immobilizable" capture probes can be contacted with the sample as well as the support.

[0193] Similarly, the primer for amplification can be immobilized before or after amplification. The capture probe can be, for example, a nucleic acid molecule capable of specifically binding to a target nucleic acid molecule. In other words, the capture probe can be a fixed (or immobilizable) probe specific for the target nucleic acid molecule, which target nucleic acid molecule contains a binding domain complementary thereto. Thus, in such an embodiment, the target nucleic acid molecule is first captured by the fixed or immobilizable capture probe, which probe is only used to immobilize the target nucleic acid molecule on the solid phase, and then the immobilized target nucleic acid molecule is subjected to a detection protocol that uses or results in the production of an amplified ligation product. More particularly, such a capture probe specifically binds the analyte.

[0194] The solid support can be any well-known support or matrix currently widely used or proposed for immobilization, separation, etc. These can take the form of particles (e.g., beads that can be magnetic or non-magnetic), sheets, gels, filters, membranes, fibers, capillaries or microtiter strips, tubes, plates or wells, etc.

[0195] The support can be made of glass, silica, latex or polymeric materials. Suitable are materials that present a high surface area for analyte binding. Such supports can have an irregular surface and can be, for example, porous or granular, such as particles, fibers, fiber meshes, sintered materials or sieves. Granular materials (e.g., beads) are useful because of their large binding capacity, especially polymeric beads.

[0196] Conveniently, the particulate solid support used according to the present invention will comprise spherical beads. The size of the beads is not critical, but they can be, for example, on 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.

[0197] Monodisperse particles, i.e., particles of substantially uniform size (e.g., a size with a standard deviation of less than 5% in diameter), have the advantage of providing very uniform reaction reproducibility. Representative monodisperse polymer particles can be produced by the techniques described in US-A-4336173.

[0198] However, for ease of manipulation and separation, magnetic beads are advantageous. As used herein, the term "magnetic" means a support that is capable of acquiring a magnetic moment (i.e., paramagnetic) when placed in a magnetic field and is thus movable under the influence of that field. In other words, supports containing magnetic particles can be easily removed by magnetic agglomeration, which provides a rapid, simple, and effective way to separate the particles after the binding step.

[0199] In another embodiment, the target nucleic acid molecule itself can be immobilized (or immobilizable) on a solid phase, for example, by non-specific absorption. In one specific such embodiment, the analyte can be present intracellularly, optionally after fixation and / or permeabilization, and the cell (capable of) attaching to a solid support, for example, a tissue sample containing the analyte can be fixed on a microscope slide.

[0200] As mentioned above, the present invention also provides certain probes for use in the methods of the present invention, namely, cyclizable probes as defined above, which can be provided in one or more parts. The present invention thus provides chimeric DNA-RNA padlock probes as defined herein, which include invasive padlock probes.

[0201] In one such embodiment, the chimeric DNA-RNA padlock probe is a single cyclizable oligonucleotide that comprises a first target-specific target-binding site located at or internal to the 5' end of the probe and a second target-specific binding site located at the 3' end of the probe, and wherein:

[0202] (i) the second target-specific binding site comprises one or more ribonucleotides;

[0203] (ii) When 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 may optionally contain one or more ribonucleotides;

[0204] (iii) The probe is mainly composed of DNA when ligated to form a ring and contains no more than 4 consecutive ribonucleotides.

[0205] In another embodiment, a chimeric DNA-RNA padlock probe comprises two or more parts, namely a first part of a backbone oligonucleotide that comprises a first target-specific target binding site located at or internal to 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 that is 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:

[0206] (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 its 3'-end;

[0207] (ii) When 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 may optionally contain one or more ribonucleotides;

[0208] (iii) One or more nicked oligonucleotides optionally comprise additional sequences 5' of the target-specific binding site such that when the nicked 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 produce a ligatable 5' end that can be ligated to the 3' end of another nicked oligonucleotide or the 3' end of the backbone oligonucleotide to circularize the probe, wherein the additional sequence can optionally contain one or more ribonucleotides.

[0209] (iv) When ligated, the backbone and nicked oligonucleotides form a loop that consists mainly of DNA and contains no more than 4 consecutive ribonucleotides.

[0210] Such probes can be invasive padlock probes, also referred to herein as iLock probes. In certain embodiments, the probes of the present invention (i.e., the probes provided as a single circularizable oligonucleotide or the portions of the probes provided as two or more parts, i.e., the backbone oligonucleotide and / or one or more nicked oligonucleotides) can thus comprise the first target-specific binding site as described herein or additional sequences 5' of the target binding site of the nicked oligonucleotide, wherein the nucleotide at the 3' end of any additional sequence forming the 5' flap is complementary to the homologous nucleotide in the target nucleic acid molecule that is the same as the nucleotide at the 3' end of the probe, or the backbone oligonucleotide and / or one or more nicked oligonucleotides (i.e., the ligatable end of the adjacent portion of the probe). When such a probe hybridizes to a target nucleic acid molecule, the nucleotide at the 3' end of the additional sequence forming the 5' flap is thus prevented from hybridizing to the target nucleic acid molecule by the 3' ligatable end of the probe or the backbone oligonucleotide and / or one or more nicked oligonucleotides.

[0211] Such invasive probes can be suitable for detecting variant bases in a target nucleic acid molecule. In probes for detecting variant bases in a target nucleic acid molecule:

[0212] (i) The nucleotide at the 3' end of the probe or the backbone oligonucleotide and / or nicked oligonucleotide as described above 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 cannot hybridize to the target nucleic acid molecule simultaneously with the 3' end of the probe or the backbone oligonucleotide or nicked oligonucleotide, such that the additional sequence can be removed by cleavage to produce a 5' ligatable end of the probe; or

[0213] (ii) The nucleotide at the 3'-end of the additional sequence and the nucleotide at the 3'-end of the probe or backbone oligonucleotide and / or nicking oligonucleotide are not complementary to the variant base, such that the nucleotide at the 3'-end of the probe, or backbone oligonucleotide and / or nicking oligonucleotide cannot hybridize to the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3'-end of the additional sequence can also be removed by cleavage.

[0214] Alternatively, in a probe for detecting a variant base in a target nucleic acid molecule:

[0215] (i) The nucleotide at the 5'-end of the first target-specific binding site or at the 5'-end of the target-specific binding site of the backbone oligonucleotide or nicking oligonucleotide is complementary to the variant base, the nucleotide at the 3'-end of the probe or backbone oligonucleotide and / or nicking oligonucleotide as described above 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 cannot hybridize to the target nucleic acid molecule simultaneously with the nucleotide at the 3'-end of the probe or backbone oligonucleotide or nicking oligonucleotide, such that the additional sequence can be removed by cleavage to generate a 5'-ligatable end of the probe; or

[0216] (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 nicking oligonucleotide is not complementary to the variant base, and the nucleotide is also removed by cleavage, thereby creating a nick between the 5'-ligatable end and the 3'-ligatable end and preventing ligation.

[0217] In a preferred embodiment, the nucleotide at the 3'-end of the additional sequence forming the 5'-flap is a ribonucleotide.

[0218] In another aspect, the present invention provides a set of invasive probes comprising two or more invasive probes for detecting a variant base in a target nucleic acid molecule, wherein each probe in the set of probes contains a different nucleotide (e.g., A, G, C, or T / U) at a position in the additional sequence and at the 3'-end of the probe or backbone oligonucleotide or nicking oligonucleotide that is complementary to the variant base. In certain embodiments, the set of invasive probes can comprise three or four probes, each probe containing a different nucleotide at said position.

[0219] As discussed above, the total number of ribonucleotides in the probe is not critical, provided that the probe contains no more than 4, or more preferably no more than 3 or 2 consecutive ribonucleotides upon ligation (see discussion above).

[0220] In some embodiments, the second binding site at the 3' end of a single-part probe or at the 3' end of the backbone oligonucleotide, or the target-specific binding site of a nicked oligonucleotide may comprise no more than 4 or 5 ribonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0222] Figure 1 Shows the role of RNA nucleotides at the 3'-linkable end at the ligation site in the ligation of RNA-templated padlock probes. A: Experimental overview. B: Padlock probes (PLPs) targeting members of the let-7 family were designed with RNA or DNA terminal 3'-nucleotides. The probes were hybridized to the matching template, ligated with 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.

[0223] Figure 2 Shows the role of RNA nucleotides at the 3'-linkable end at the ligation site using PBCV-1 or T4Rnl2 ligase. The ligation using RNA template, 3'-OH(N) / 5'-p(N) was compared to 3'-OH(rN) / 5'-p(N). Intact DNA and chimeric padlock probes were hybridized to the corresponding RNA targets and ligated with (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 contains 3'-ribonucleotides at its 3' end, a larger number of ligation products were seen for all target RNAs with PBCV-1 and a substantial increase in the number of ligation products was seen for all target RNAs with the T4RnI2 ligase.

[0224] Figure 3 Shows the effect of 3'-OH(rN) mismatches on nick sealing by PBCV-1 and T4Rnl2 ligases. The number of RCPs for each RNA template ( Figure 3 B and 3C) was summed and expressed as a percentage within the iLock probe set for each ligase ( Figure 3 A).

[0225] Figure 4 Shows the role of RNA substitutions at various positions in the invasive padlock (iLock) probe in an RNA detection assay using the PBCV-1 ligase. The recognition of the invasive structure and structure-specific nicking 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 and the arrangement of the 5' appended sequence. RNA nucleotides were introduced at different positions: at the terminal 3' end (3); the 3'-most distal nucleotide in the 5' flap that competes with the terminal 3' nucleotide at the probe end for target binding (displaced base, D); the base at the first target-binding site that becomes the 5'-ligatable end (providing a 5'-phosphorylated donor) after cleavage to remove the appended sequence (iLock probe activation) (5); the entire flap sequence (F). B: The cyclization of six iLock designs was evaluated: DNA-only iLock; iLock with (3) modification (iLock-3); iLock with (3) and (D) modifications (iLock-3D); iLock with (3), (D), and (5) modifications (iLock-3D5); iLock with (3), (D), and (F) modifications (iLock-3DF); and iLock with (D) and (F) modifications (iLock-DF). The total number of RCPs detected for each iLock probe is shown on the x-axis. Probes containing the (3) modification, and combinations of the (3) modification with (D) or (D) and (F) modifications, showed a substantial increase in the number of RCPs produced compared to the DNA-only iLock. Combinations of the (3) modification with (D) and (5) modifications, and (D) and (F) modifications, showed a much smaller increase in the relative number of RCPs produced compared to the DNA-only iLock. C: PAGE of iLock DNA, iLock-3, and iLock-3D probes after activation and ligation in the absence (lanes 1-3) and presence (lanes 4-6) of Taq DNA polymerase. The unactivated iLock probe (79) is shortened by 14 nt (65) after activation and ligated (see the high molecular weight band at the top of the gel). Lane 4: Bands of the activated unligated probe are 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 unligated 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 unligated probe is visible, the band of the uncleaved probe is faint (65 nt), and a strong band of the ligated probe is visible. Together with these data, it is shown that ribonuclease at the 3'-ligatable end at the ligation site improves ligation (lanes 4 and 5), and ribonuclease at the most 3'-terminal position in the appended sequence that is cleaved in the invasion assay improves cleavage (lanes 5 and 6).

[0226] Figure 5 Shows a comparison of chimeric and non-chimeric iLock probe ligation. A: Performance of 3D and non-chimeric iLock probes on longer non-miRNA targets. The total number of RCPs for each probe on the matched polymorphic template 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 the chimeric or non-chimeric iLock probe is presented on the y-axis. The ligases used are depicted on the x-axis. Error bars ± standard error; n = 2. When using chimeric probes, both ligases showed improved ligation.

[0227] Figure 6 Shows the ligation efficiency and fidelity of chimeric iLock probes on non-miRNA templates for PBCV-1 and T4Rnl2 ligases. A and B: Fidelity of nick-sealing by PBCV-1 and T4Rnl2 ligases on the matched polymorphic RNA template. C and D: Data presented in (A) and (B), but presented as the total number of RCPs generated for each iLock probe on each polymorphic template. Error bars ± standard error; n = 2.

[0228] Figure 7 Shows multiplex detection of let-7 miRNA isoforms using chimeric iLock probes and PBCV-1 ligase. 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 pool of sequencing library oligonucleotides competes for hybridization based on the nucleotide at their 5'-end. Libraries containing a terminal T are 3'-FITC labeled; G - 3' Cy3 labeled; A - 3' Cy5 labeled. The ligase joins the library oligonucleotide corresponding to the barcode base. B: Imaging of the first barcode base by Sequencing by Ligation (SBL). AP: All RCPs stained with AP. Images of each barcode base and the merged image are presented. Error bars 5μm. C: miRNAs are mixed in the stoichiometric ratios stated on the x-axis. 1:1:1 represents equal ratios and 0:0:0 represents 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 produces a similar number of RCPs.

[0229] Figure 8 Shows 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 RNl2 ligase using (A) RNA templates or (B) DNA templates at high and low concentrations. For both ligases, for RNA and DNA targets, chimeric padlock probes showed more efficient ligation and amplification than DNA-only padlock probes.

[0230] Figure 9 Shows in situ detection of KRAS wt and mutant RNA using DNA padlock probes or chimeric padlock probes. A: Microscopic images showing the 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 chimeric padlock probes was much higher. Specificity was high enough to distinguish mutant and wild-type KRAS (more mutant RCPs in A549 and more wild-type RCPs in OncoDG1).

[0231] Figure 10 Shows in situ detection of KRAS wt and mutant RNA using chimeric iLock probes. A: Microscopic images showing the detection of mutant and WT RNA using chimeric iLock probes. B: Average number of mutant RCPs and wild-type RCPs per cell in cell lines A549 and OncoDG1. Specificity was high enough to distinguish mutant and wild-type KRAS (more mutant RCPs in A549 and more wild-type RCPs in OncoDG1).

[0232] Figure 11 Shows the detection of target RNA molecules using gap-filling polymerization and iLock probes. A: Number of RCPs counted in solution after reverse transcriptase gap-filling polymerization and Taq cleavage + PBCV-1 ligase ligation (both in one step) followed by RCA using chimeric iLock probes. B: In situ detection of RCPs using gap-filling polymerization and chimeric iLock probes.

[0233] Figure 12 Shows the design of a two-part iLock probe that contains ribonucleotides at the ends of the backbone and the nicking oligonucleotide. The target RNA molecule (1) is contacted with a two-part iLock probe containing a backbone oligonucleotide (2) and a gap-filling oligonucleotide (3). Both the backbone oligonucleotide and the gap-filling oligonucleotide contain additional sequences (4) at their 5'-ends that do not hybridize to the target RNA molecule and ribonucleotides (5) at their 3'-ends.

[0234] Figure 13 Shows the effect of RNA substitution on rolling circle amplification using Phi29 DNA polymerase. A: Total RCA produced by padlock probes (y-axis) in the presence / absence of terminal 3' RNA and in the absence of synthetic RNA ligation template (template-). B: Loops with 0 - 7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCP); error bars ± standard error; n = 2. The same RCA reactions with chimeric loops were also monitored by real-time measurement of Sybr Gold incorporation on a qPCR instrument (C and E). C: RCA reaction curves for loops with 0, 1, 2, or 3 RNA substituents. 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 within 1.5 of the hinge quartile range; lower whisker, minimum within 1.5 of the hinge quartile range. E: Shows real-time data for RCA reactions with 0 - 7 RNA substituents as in B. Representative samples are presented from replicate experiments. To highlight the initial phase of RCA and to see differences between samples with low RCA efficiency, fluorescence between 4000 and 6000 is shown.

[0235] Figure 14 Show , compared to padlock probes containing deoxyribonucleotides at their 3' ends, chimeric padlock probes containing 3' ribonucleotides are more easily ligated using PBCV-1 ligase. Lanes 1 - 6 - chimeric padlock probes Lanes 7 - 12 - non-chimeric padlock probes The ligated probe products are shown as heavier fragments ( * ). For chimeric probes (lanes 2 - 3), this is clearly visible after 1 - 2 minutes, while for non-chimeric probes (lane 12), it is only clearly visible at a later time point.

[0236] Figure 15 Shows a nicked-fill padlock probe (2) containing ribonucleotides (3) at the 3' end of a 5' appended sequence (4), which is cleaved prior to ligation. The 3' end (5) of the probe hybridized to the target nucleic acid molecule (1) can be extended by nick-fill polymerization.

[0237] Figure 16 Shows a padlock probe (2) that contains ribonucleotides (3) at positions at or near the ligation site (4) other than the site where it hybridizes to the target nucleic acid molecule (1).

[0238] Figure 17 Shows a padlock probe (2) that includes a backbone oligonucleotide provided in two parts, and a ligation template (4) that 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.

[0239] Figure 18 The amplification rates of circular padlock probes containing different ribonucleotides are shown. A: Real-time RCA curves of loops with 1, 2, or 3 consecutive RNA substitutions containing all four RNA bases are shown. The rate of RCA was monitored by measuring the fluorescence accumulation (y-axis) generated by SybrGold incorporation into the RCP. Representative data for each experiment are shown. B: The RCA rates of a positive control (pure DNA loop - lower left panel), a negative control (no loop), and loops with 2, 3, 5, and 7 consecutive RNA substitutions, as well as loops with RNA substitutions scattered between DNA bases, are shown. Phi29 DNA polymerase exhibited a higher RCA rate using loops with pyrimidine RNA substitutions.

[0240] Figure 19 It is shown that limited replication of RNA-rich padlock probes was not recovered in the presence of M-MuLV reverse transcriptase. Amplification curves of padlock probes with 0 - 7 RNA substitutions in the backbone are shown. The rate of RCA was monitored by measuring the fluorescence accumulation (y-axis; 3000–30000) generated by SybrGold incorporation into the RCP. Replication of loops without additional reverse transcriptase (upper panel) and with additional M-MuLV reverse transcriptase (lower panel) is shown.

[0241] Figure 20 Stacked plots showing the incorporation of the expected dNTPs during RCA reverse transcription are shown. RNA-containing padlock probes were amplified, monomerized, and sequenced. RCA monomers were generated from a control DNA loop (upper row), and loops containing rA, rC, rG, and rU at the first RNA position (R1), and rUrU, rArA, rCrC, and rGrG at their R1 and R2 positions (complete oligonucleotides are in Table 9). Sequencing reads were aligned and the frequency of each base at each position was calculated. The size of each base is proportional to the base frequency. Positions R1 and R2 (relative to the RNA positions in the padlock probe backbone) are indicated by boxes and highlight position R1 (see arrow).

[0242] Figure 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 padlock probes (PLP) and iLock probes. Probes for both targets were included in each sample, and a good level of target specificity was shown. B: Using chimeric and DNA-only PLP and iLock, the average number of RCPs per cell produced by each probe for each cell line is shown. 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). The mouse-specific PLP and PLPr show no signal. For the iLock (third and fourth box plots), RiLock shows a median higher than iLock (box plots slightly shifted), but the signal amount 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 the signal from the human-specific probe.

[0243] Figure 22 In situ detection of miR21 RNA immobilized on a solid surface is shown. A: miR21 is immobilized and hybridized with complementary probes (labeled with a fluorescent dye). The edges of the silicone chamber were 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 quantified number of RCPs is expressed as the total number of RCPs / field of view (FOV).

[0244] Figure 23 In situ multiplex RNA detection using chimeric padlock probes and in situ sequencing in mouse brain tissue sections are shown. The upper panel shows an overview image of a mouse brain tissue section, where the nuclei are stained with DAPI and the RCA products stained with the anchor probe are produced by chimeric PLP targeting 18 different neuronal genes (5 probes per gene, each = a total of 90 different probes). Below is the area where individual cells are visible in the left overview image.

[0245] Figure 24 It is shown that with a loop containing pyrimidine RNA substitutions, Phi29 DNA polymerase exhibits a higher RCA rate. (A) shows real-time RCA curves of loops with 1, 2, 3, or 4 consecutive RNA substitutions containing rG, rU, rA, rC RNA bases (the number of consecutive substitutions is indicated as in the curves). 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. RCA was carried out in the presence of Mg 2+ and Mn 2+ (solid and dashed lines, respectively). (B) presents the linear, early RCA velocities (y-axis) of the PLP from (A) in the presence of Mg 2+ (solid line) and Mn 2+ (dashed line). (C) shows the RCA of a control PLP (non-chimeric DNA loop) in the presence of Mg 2+ (solid line) and Mn 2+ (dashed line).

[0246] Figure 25 It is shown the RCA of chimeric circular substrates with RNA substitutions organized in different patterns. The rate of RCA was monitored by measuring the fluorescence accumulation (y-axis) generated by SybrGold incorporation into RCP. Four, five, and seven consecutive substitutions (yellow, red, green); three, six RNA substitutions (blue, gray) scattered among one or two DNA bases, and three and eight RNA substitutions (orange, magenta) (only legend) scattered among a large number of DNA bases were introduced into the PLP backbone as shown in the inset (only a fragment depicting the backbone fragment, the complete PLP sequence as indicated). Average data from repeated experiments. RCA was carried out in the presence of magnesium and manganese ions (solid and dashed lines, respectively).

[0247] Figure 26 Show It shows the effects of RNA substitutions on the stability of 3'-OH (rG) and 3'-OH (G) padlock probes and on the ligation with PBCV-1 ligase on RNA. A: PBCV-1 ligase titration. The total number of RCPs (y-axis) generated at each ligase concentration (x-axis) during a 30-min ligation. For each time point, data for chimeric probes are shown on the left and data for non-chimeric probes are shown on the right. B: To evaluate the stability of 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.

[0248] Figure 27 Shows a comparison of miR21 and let-7f using PBCV-1 and T4Rnl2, chimeric and non-chimeric iLock probe ligation. 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.

[0249] Figure 28 Shows the effect of 3'-OH(rN) mismatches on iLock activation and the nick-sealing fidelity of PBCV-1 and T4Rnl2 ligases. A and B: Performance of chimeric iLock-3D (left) and non-chimeric (right) iLock probes on polymorphic RNA targets. For A) PBCV-1 DNA ligase and B) T4Rnl2, the total number of RCPs for each probe on the matching polymorphic template is shown on the y-axis. NC - negative control. C: Nick-sealing fidelity using 3D-type iLock probes on RNA by PBCV-1 DNA ligase (left panel) and T4Rnl2 (right panel). The number of RCPs for the same iLock probe on each RNA template is summed and presented as a percentage within the iLock probe group. The calculated ratio of the expected probe pairs is highlighted.

[0250] Figure 29 Shows the ligation efficiency and fidelity of chimeric iLock probes with PBCV-1 and T4Rnl2 ligases on polymorphic templates. For A) PBCV-1 DNA ligase and B) T4Rnl2, the total number of RCPs generated and quantified for each iLock probe on each polymorphic template (y-axis) is shown Figure 28 in.

[0251] Figure 30 Shows dNTP incorporation during RCA reverse transcription. As described in the examples, padlock probes are monomerized, amplified, and sequenced. As depicted in the following separate figures, samples with single and double RNA bases are sequenced using this method. Sequencing reads are aligned and the frequency of each base at each position is calculated. 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 positions of RNA bases are indicated by boxes. The DNA / RNA sequence initially presented in the PLP sequence is depicted on the right.

[0252] Figure 31 Shows the error incorporation rate at each position in the padlock probe backbone during RCA reverse transcription. The probability of an unexpected nucleotide mismatch at each position for padlock probes with single (left panel) and double RNA substitutions (right panel) was calculated as incorporation error [%] = 1 - reads of expected nucleotide / total reads. Shown is each base of the sequencing reads (x-axis) and the average error (y-axis) for each analyzed sample. The RCA reaction was carried out in the presence of manganese (A) and magnesium (B).

[0253] Figure 32 Shows the effect of RNA substitution in the circular template on rolling circle amplification using phi29 DNA polymerase. (A) Circles with 0 - 7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± standard error; n = 2. The same RCA reaction with chimeric circles was also monitored in real-time by measuring SYBR gold incorporation on a qPCR instrument (B and C). (B) RCA reaction curves for circles with 0, 1, and 2 RNA substitutions. (C) Shows real-time data for the RCA reaction with 0 - 7 RNA substituents, the same as in B. Representative samples are presented from repeated experiments. To highlight the initial phase of RCA and show the differences between samples with low RCA efficiency, fluorescence intensity readouts between 3000 and 6000 are presented.

[0254] Figure 33 Shows DNA sequencing-based rolling circle product analysis, revealing the reverse transcription activity of phi29 DNA polymerase. (A) After RCA, short DNA oligonucleotides were hybridized to the AluI restriction sites in the RCA products, and the RCPs were digested with AluI restriction enzyme to obtain RCA monomers. After digestion, the monomers were PCR amplified using primers containing Ilumina adapter sequences. The PCR products were extended using Ilumina index primers. Finally, a sequencing library was prepared using index primer-specific P5 / 7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated by a green box. (B) Logo showing the sequencing frequency at each position in the RCA monomers generated from a control DNA circle (P1 = dG), and circles containing single rG, rU, rA, and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 is highlighted with a red box. (C) Incorrect nucleotide incorporation at each position in the sequenced monomers from (B). The error rates for padlock probes with single (upper panel) and double RNA substitutions (lower panel) are presented, calculated as incorporation error [%] = 1 - number of reads with expected nucleotide / total number of reads. The P1 position of the first RNA substitution is boxed. Detailed Description

[0255] Example

[0256] Example 1 - Detection of miRNA Using a Chimeric DNA / RNA iLock Probe and the New Activity of PBCV-1 DNA Ligase: RNA Template-Dependent Ligation Figure 4

[0257] Materials and Methods

[0258] Oligonucleotides Used in This Study

[0259] All oligonucleotides used were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA) and were synthesized and purified using the following conditions: DNA padlock probes and iLock probes: 4 nM standard desalted DNA oligonucleotides; chimeric padlock and iLock probes: 4 nM standard desalted RNA oligonucleotides; adornment 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 positioned polymorphic sites are shown in Table 1 (benchmark oligonucleotides), where the polymorphisms are indicated by asterisks.

[0260] Padlock probes were designed such that when the bases pair with the participating RNA target, the terminal arms will form a nicked loop and the discriminator base is located at the 3'-end of the probe (Table 1). miRNA padlock probes used in this study are shown in Table 1. Chimeric padlock probes are sorted by terminal 3'-OH RNA. iLock probes were used in this work for comparison purposes (Table 2). The standardized chimeric iLock probe design includes the terminal 3'-base and bases in the 5'-arm that compete with the 3'-terminal base for target binding (replacement bases, Figure 14 A, Table 2) with RNA substitutions. Two types of probe barcoding methods were used: traditional and compatible with ligation sequencing readout (for chimeric, miRNA-targeting iLock probes). For traditional rolling circle product (RCP) staining and digital quantification, a reporter sequence was embedded in the sequence of the ligation 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 anchored primer sequence was embedded in the probe backbone, followed by a two-base barcode and a sequencing library of the anchored sequence (Table 3).

[0261] RNA Detection Assay and Digital Quantification of Amplified iLock and Padlock Probes

[0262] iLock activation (cleavage) was carried out with a 4:1 probe-to-template excess (typically, 2 nM iLock probe was mixed with 0.5 nM RNA template). The reaction in duplicate was incubated at 51 °C for 30 min in a heated-lid thermal cycler in a 10 μL volume containing 1 U Taq DNA polymerase (ThermoFisher Scientific), 4 U RNase inhibitor, and 1× Taq polymerase buffer supplemented with 8 mM MgCl2. Subsequently, 3 μL of the 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 T4 Rnl2 (M0239S, NEB) in their respective buffers, with a final volume of 15 μL. The reaction was stirred at 37 °C for 30 min. For padlock probes, the same ligation conditions were applied, excluding the activation step. For RCA, 5 μL of the ligation reaction was incubated with 10 nmol of the adornment probe, 0.125 mM dNTP, 0.2 μg / μl BSA, 250 mU Phi29 polymerase (Monserate Biotechnology Group), and 1× phi29 reaction buffer (Thermo Fisher) at 37 °C for 60 min in a final volume of 25 μl. The polymerase was heat-inactivated at 65 °C for 3 min and cooled to room temperature. Unless otherwise stated, the estimated final concentrations of the amplification products for padlock probes and iLock probes were 5 pM and 20 pM, respectively. A 15 μl RCA sample was analyzed using an Aquila 400 detection unit (Q-linea, Uppsala). If the RCP concentration was outside the dynamic range of the instrument, the sample was diluted in 4 nM adornment 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, cooled to room temperature for 15 min, and re-counted. A template-negative reaction was carried out in parallel with each experiment as a control.

[0263] Multiplex miRNA detection using chimeric iLock probes and ligation sequencing

[0264] To test whether chimeric iLock probes can be used to detect miRNA expression changes in RNA mixtures, we have combined (let-7f):(let-7e):(let-7d) miRNAs at (1):(1):(1), (3):(1):(1), (1):(3):(1) and (1):(1):(3) ratios. The baseline miRNA concentration during the iLock activation step was 0.5 nM, while the baseline miRNA concentration of samples with elevated miRNA concentration was 1.5 nM. A mixture of 2 nM let-7f, let-7e, let-7d chimeric iLock probes, each probe embedded with a unique two-nucleotide barcode (Table 3) and a ligation sequencing chemical sequence, was used. The experiment was conducted as described above (using PBCV-1 ligase), except that 10 μL of the RCA product was spotted onto a positively charged microscope slide (Superfrost Plus, Menzel ) and evaporated at 55 °C for 10 min. A 50 μL volume of silicone chamber (Secure-Seal hybridization chamber, Sigma) was placed over each droplet, and the samples were washed 3 times with 1×TBS. 0.5 μM of the anchored primer was hybridized in 2×SSC, 20% formamide for 30 min at room temperature. After washing 3 times with 1×TBS, the RCP was mixed with the sequencing mixture containing 1×T4 DNA ligase buffer, 10 μg BSA, 1 mM ATP, 0.1 μM sequencing oligonucleotide (Table 3) and 5 U 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 antifade reagent, ThermoFisher). Images of the RCP were acquired using a 20× objective lens.

[0265] 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 probes were processed as above. After ligation, 50 nM of the sample was diluted to a final volume of 12 μL in TBE-urea sample buffer (LC6876, ThermoFisher Scientific). The sample was denatured at 70 °C for 3 min, placed on ice for 5 min, and 10 μL was loaded onto a 15% TBE-urea gel (EC6885BOX, ThermoFisher Scientific) and run in an XCell SureLock TMIn a Mini - Sub Cell GT electrophoresis system (ThemoFisher Scientific), a PowerPac Basic power supply (Bio - Rad) was used to separate for approximately 90 min at 170 V. The gel was stained with 1×SybrGold (S11194, Invitrogen) in 1×TBE running buffer for 15 min and then imaged in a Gel Doc XR System (Bio - Rad). In the chimeric probe ligation assay, a certain concentration of template and padlock probe as stated above were incubated with 62 mU / μL PBCV - 1 DNA ligase and 0.4 U / μL RNase inhibitor for 10 min at room temperature. The reaction was terminated by adding 1 μL of 0.5 M EDTA and 5 μL of 100% formamide.

[0266] Results

[0267] Effect of 3'-OH RNA on RNA - dependent ligation by PBCV - 1 DNA ligase: RNA end joining for different RNA substrates

[0268] We compared the ability of PBCV - 1 ligase to circularize padlock probes hybridized to let - 7a, where the 5'-end of the probe was DNA and the 3'-end was either DNA or RNA. Although the ability of T4Rnl2 to join a chimeric 3'-RNA acceptor strand to a 5'-donor strand on RNA has been well - characterized, only PBCV - 1 DNA ligase on a DNA template demonstrated this. By PAGE separation, we compared the ligation efficiency of chimeric padlock probes relative to DNA padlock probes over the reaction time ( Figure 1 ). Additionally, we measured the ligation efficiency as the total number of rolling circle amplification products (RCPs), counting the digital number for each padlock / template pair ( Figure 1 A).

[0269] When the PLP containing 3'-RNA was ligated to the miRNA target, PBCV - 1 ligase catalyzed efficient end joining ( Figure 2 B). For longer non - miRNA targets, the ligation efficiency of chimeric and non - chimeric probes was similar ( Figure 2 A). Since the presence of RNA results in greater nucleic acid duplex stability, we hypothesized that more stable duplexes would be ligated faster during the initial reaction phase. T4Rnl2 efficiently ligated chimeric padlock probes, while the activity of DNA padlock probes was relatively low ( Figure 3 B). We found that PBCV - 1 readily accepted chimeric padlock probes as substrates, which prompted us to systematically characterize the ligation fidelity on synthetic targets with polymorphic positions in the centrally - located nucleotides ( Figure 3and Table 1). To measure the effect of mismatched chimeric substrates on the end-joining activity of PBCV-1 and T4Rnl2 ligases, four chimeric padlock probes with different 3'-terminal nucleotides (rA, rU, rG, rC) were each hybridized to four different RNA targets, ligated, and amplified by RCA ( Figure 3 ). The PBCV-1 ligase is highly tolerant to most 3'-RNA mismatches ( Figure 3 A, 3B). On the other hand, T4Rnl2 ligates rC / rG (82%) and rA / rC (64%) with moderate accuracy but has poor end-joining fidelity for other combinations ( Figure 4 A, 3C).

[0270] Effects of various RNA substitutions on RNA-templated iLock probe activation, ligation efficiency, and fidelity of PBCV-1 and T4Rnl2

[0271] In our previous study, we utilized 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 ligation-based RNA detection fidelity (Krzywkowski, see above). Since the PBCV-1 ligase is quite tolerant to most of the 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 DNA iLock probes. Multiple iLock probes targeting let-7a miRNA were designed (Table 2), which contained RNA substitutions in various probe positions ( Figure 4 A). One chimeric probe (referred to as "3") had an RNA substitution at the 3'-end. In the "3D" probe, the displaced bases of the terminal 3'- and 5'-flaps were replaced with RNA. The "3D5" probe had an additional RNA base at position 3' of the "D" position in addition to the substitutions in the "3D" probe. After successful activation of iLock, this RNA base would become the 5'-phosphate donor end in the ligation reaction. Finally, we designed probes with terminal 3'- and full 5'-flaps as RNA bases ("3DF"), and probes in which only the 5'-flap was composed of RNA ("DF") (i.e., lacking 3'-RNA).

[0272] Compared to non-chimeric let-7a iLock, iLock-3 significantly increased the detection of let-7a miRNA. According to the PAGE of ligated iLock probes ( Figure 4 C), under the given conditions, only a small fraction of non-chimeric iLock probes were activated (cleaved), and an even smaller fraction was ligated ( Figure 4C, lane 4). Almost all of the activated iLock-3 probes have been ligated, as Figure 4 quantitative gel shift in C (lane 5) and the total number of RCA products generated by the iLock-3 probe Figure 4 B). When the flap nucleotide replaced by the invading 3'-terminal RNA was replaced by RNA, as in iLock-3D, an additional efficiency increase was observed. Most of the iLock-3D was activated and ligated Figure 4 C). For the iLock-3DF probe, a similar effect was observed, where the entire 5'-flap sequence was RNA and the positive effect was lost in the absence of the terminal 3'-RNA base Figure 5 B). Interestingly, the iLock-3D5 probe containing 3'-(rN) / 5'-(rN) after activation showed significantly lower performance than the iLock probe with deoxyribonucleotide at the (5) position. For other miRNAs (miR21) tested using PBCV-1 and T4Rnl2 ligases, a significant performance improvement of the iLock-3D probe was observed Figure 27 ). Similarly, in repeated experiments using other iLock-3D probes, an improvement in performance was also observed for PBCV-1 and T4Rnl2 using let-7f as a template Figure 6 ). To test whether the accuracy of RNA sensing was maintained using chimeric iLock-3D probes, we targeted four polymorphic RNA templates using four chimeric iLock-3D probes (Table 2). The chimeric iLock probes showed excellent fidelity against the matching rC / rG, rA / rU, and rU / rA probe pairs Figure 6 ). When the template was omitted, no ligation products were shown by the iLock-3D probe. T4Rnl2 showed complete compatibility with the iLock RNA detection assay, thus easily ligating the target-matched 3'-OH(rN) / 5'-p(N)iLock probe Figure 6 B). For PBCV-1 and T4RNl2, the rG / rC pair was detected with relatively low fidelity, showing rG / rU misligations of 5% and 27% respectively Figure 28 A, 6B). In another experiment, both PBCV-1 and T4Rnl2 showed complete compatibility with the iLock RNA detection assay, thus easily ligating the target-matched 3'-OH(rN) / 5'-p(n)iLock probe Figure 29 A-B, Figure 28 , A-B). For both enzymes, the rG / rC pair was detected with relatively low fidelity, showing rG / rU misligations of 21% and 11% respectively. Thus, as can be seen from Figure 7As can be seen from A-B, the chimeric iLock has better performance than DNA iLock.

[0273] Multiplex detection of let-7 isoforms using chimeric iLock probes

[0274] High multiplexing ability is one of the most advantageous features of padlock probes. Discrimination of amplification products derived from different padlock probes is typically achieved by using unique probe-specific adornment oligonucleotides (labeled with fluorophores having different emission spectra). Alternatively, unique barcode sequences can be embedded into the padlock probe backbone, which can be decoded using next-generation ligation sequencing chemistries. To evaluate the compatibility of chimeric iLock probes with ligation sequencing readout, we redesigned four let-7 family iLock-3D probes as described in the Methods section (Table 3). To evaluate whether barcoded iLock-3D probes can be used for multiplex miRNA analysis, we combined let-7f, let-7e, let-7d miRNAs in four different stoichiometric ratios. Ideally, the ratios would be accurately reflected in miRNA-specific sequencing reads. The iLock probes were multiplexed and the amplified products were immobilized on a glass surface. The barcodes of the RCPs were decoded using ligation sequencing chemistry. Since only three iLock probes were used in this experiment, it was sufficient to sequence the first barcode position to decode the detected miRNAs. The iLock probes showed similar relative efficiencies for the miRNA pool ( Figure 7 ). In samples with increasing concentration of one miRNA, the signal of the corresponding iLock probe increased while the signals of other targets remained stable ( Example 2 – Ligation of DNA Padlock Probes and Chimeric Probes Containing 1 or 2 Ribonucleotides at Their 3' End C).

[0275] Figure 8

[0276] Materials and Methods

[0277] Ligation reactions were carried out using DNA padlock probes at a final concentration of 1 nM or chimeric padlock probes with 1 or 2 terminal 3'-ribonucleotide bases, and synthetic KRAS RNA templates or KRAS DNA templates at a final concentration of 2 nM. The oligonucleotide sequences are shown in Table 6. The reactions were incubated at 37 °C for 30 min in a final volume of 10 μL in ligation buffers 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. Subsequently, the circles were amplified by rolling circle amplification in RCA reaction buffer to a final circle concentration of 100 pM. Finally, the RCA products were labeled with Cy3-labeled detection probes at a final concentration of 10 pM. The labeled RCA products were digitally counted.

[0278] Results

[0279] For both SplintR ligase and T4 RNA ligase II, and on both RNA and DNA templates, two chimeric padlock probes with 1 or 2 terminal 3'-ribonucleotide bases produced more RCA products than the pure DNA padlock probes ( Figure 8 ). Increasing the ligase concentration had no effect on the chimeric padlock probes but had a slight negative effect on the DNA padlock probes. There was no difference in the RCA product counts between the chimeric padlock probes with 1 or 2 terminal 3'-ribonucleotide bases.

[0280] On the RNA template, the activities of SplintR ligase and T4 RNA ligase II were similar ( Figure 8 A). On the DNA template, for SplintR ligase, the difference between the DNA padlock probe and the chimeric padlock probe was similar to that on the RNA template, but a very strong increase in RCP counts was recorded for the ligation of chimeric padlock probes on the DNA template using T4 RNA ligase II ( Example 3 – In Situ KRAS Point Mutation Detection Using Chimeric Padlock Probes and Chimeric iLock Probes B). T4 RNA ligase II does not accept 3 ' DNA and 5 ' DNA ends when templated by DNA, but readily accepts probes with 3 ' RNA ends when templated by DNA.

[0281] In summary, the use of chimeric probes made the ligation reactions on RNA templates using SplintR ligase and T4 RNA ligase II more efficient than with conventional DNA probes. The chimeric probes enabled T4 RNA ligase II to be used for ligation reactions on DNA templates.

[0282] Figure 9

[0283] Materials and Methods

[0284] The ONCO-DG-1 and A-427 cell lines were cultured in RPMI medium without L-glutamine supplemented with 10% FBS, 2 mM L-glutamine, and 1x penicillin-streptomycin (PEST). The A-549 was cultured in DMEM supplemented with 10% FBS and 1x PEST. At confluence, all cell lines were seeded on Superfrost Plus slides and allowed to attach for 12 h. Then the cells were fixed in 3% paraformaldehyde in DEPC-treated PBS (DEPC-PBS) for 15 min at room temperature. After fixation, the slides were washed twice in DEPC-PBS and dehydrated through a series of 70%, 85%, and 100% ethanol for 4 min each. A coverslip was mounted on the slides, and the cells were hydrated by a brief wash with PBS-T (DEPC-PBS with 0.05% Tween 20), then permeabilized with 0.1 HCl in H2O for 1 min at room temperature. The cells were washed twice in DEPC-PBS-T, and then DNA or chimeric probes were added to the hybridization buffer containing 2x SSC, 20% formamide, and 0.4 U / μL RNase inhibitor at a final concentration of 50 nM. The oligonucleotide sequences are shown in Table 6. The probes were hybridized at 37 °C for 60 min. Then the probe hybridization mixture was removed and the cells were washed at 37 °C for 15 min in pre-warmed (37 °C) wash buffer containing 2x SSC and 25% formamide, and washed once at 37 °C for 15 min in pre-warmed (37 °C) wash buffer containing 2x SSC and 20% formamide. The cells were washed once in PBS-T. Then the ligation reaction mixture was added to the DNA / chimeric padlock probe experiment ( Figure 10 ) and the invasion reaction mixture was added to the DNA / chimeric iLock experiment ( Figure 9)。The ligation reaction mixture contained SplintR ligase buffer, 0.2 mg / mL BSA, 0.8 U / μL RNase inhibitor, and 0.25 U / μL SplintR ligase. The ligation reaction was incubated at 37 °C for 60 min. The invasion reaction mixture contained the same as the ligation reaction and an additional 0.1 U / μL Taq DNA polymerase. The invasion reaction mixture was incubated at 37 °C for 60 min. Subsequently, all experimental reactions were washed twice with PBS-T. 100 nM RCA primer was in situ hybridized for 30 min at room temperature in 2x SSC and 20% formamide hybridization buffer. The cells were washed twice in PBS-T. Next, an RCA reaction mixture containing 1x phi29 reaction buffer, 0.25 mM dNTP, 0.2 mg / mL BSA, 1 U / μL phi29 polymerase, and 5% glycerol was added and incubated at 37 °C for 3 h. Subsequently, the cells were washed twice in PBS-T, and the detection probe was in situ hybridized with the RCA product (Cy3-labeled probe with KRAS wild-type probe, Cy5-labeled probe with KRAS mutant probe) 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 more times, and then fixed in Slowfade fixation medium. The cells were imaged at 20x objective on a fluorescence microscope and the RCA products were quantified using Cell profiler software.

[0285] Results

[0286] Compared with DNA padlock probes, chimeric padlock probes had higher in situ RNA detection efficiency for both A549 and OncoDG1 cell lines ( Figure 10 B). In addition, compared with DNA padlock probes, the ratio of specific / non-specific RCP per cell increased in both A549 and OncoDG1 for chimeric padlock probes (more mutant RCPs were detected than wild-type RCPs in A549 cells (carrying a KRAS codon 12 point mutation), and more wild-type RCPs were detected than mutant RCPs in OncoDG1 cells (KRAS wild-type)), which made it possible to more precisely detect point mutations in situ directly on RNA using chimeric probes.

[0287] To further improve the specificity of point mutations, we applied chimeric iLock probes in situ and found that KRAS wild-type mRNA was specifically detected in OncoDG1 cells and the KRAS codon 12 point mutation was detected in A549 cells ( Example 4 - Nick-Filling iLock Probes ).

[0288] In summary, the chimeric probes significantly improve the efficiency of in situ RNA detection, making in situ RNA analysis more sensitive, cost- and time-efficient than classical cDNA methods. Additionally, compared to DNA padlock probes, chimeric padlock probes and especially chimeric iLock probes show higher specificity, making in situ RNA analysis more effective and accurate.

[0289] Figure 11

[0290] Materials and Methods

[0291] In-solution gap-filling iLock reaction:

[0292] The ligation reaction was carried out with a final concentration of 10 nM of the gap-filling ILock probe and a final concentration of 30 nM of the synthetic KRAS RNA template (or no template in the negative control). The oligonucleotide sequences are shown in Table 7. The reaction was carried out in a reaction buffer containing 1 U / μL of RNase inhibitor, 1 U / μL of reverse transcriptase, 25 μM dNTP (or no dNTP 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 at a final volume of 10 μL for 60 min at 37 °C. Thereafter, the circles were diluted 10-fold in PBS-T (to a theoretical concentration of 1 nM) and then amplified by rolling circle amplification in the RCA reaction buffer to a final circle concentration of 100 pM. Finally, the RCA products were labeled with a Cy3-labeled detection probe at a final concentration of 10 pM and digitally counted.

[0293] In situ gap-filling iLock reaction

[0294] Prepare and process OncoDG1 cells as described in Example 3. Under the same conditions as in Example 3, hybridize the gap-filled iLock probe with KRAS RNA in situ at a concentration of 50 nM. After washing, add the gap-filled polymerization invasion mixture to the cells containing 1 U / μL RNase inhibitor, 10 U / μL reverse transcriptase, 25 μM dNTP (or no dNTP in the gap-filled 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 incubate on the cells at 37 °C for 60 min. Wash the cells twice in PBS-T. Then, add the RCA reaction mixture containing 1x phi29 reaction buffer, 0.25 mM dNTP, 0.2 mg / mL BSA, 1 U / μL phi29 polymerase, and 5% glycerol and incubate at 37 °C for 3 hours. Subsequently, wash the cells twice in PBS-T and hybridize the Cy3-labeled detection probe with the RCA product as described above. Wash the cells three times in PBS-T, stain the cell nuclei with DAPI, wash three times again, and then fix in Slowfade fixation medium. Image the cells and RCA products under a fluorescence microscope with a 20x objective and quantify the RCA products using Cell profiler software.

[0295] Results

[0296] Quantify and plot the RCA products of the solution-phase gap-filled polymerization-invasion reaction in Figure 11 A. When an RNA template is present, count the RCA products, indicating that the reaction proceeds fully. When no template (template negative) is present, a significantly lower number of RCPs are counted, indicating that the gap-filled iLock probe cannot be extended and thus the 5 ' flap cannot be removed and cannot be ligated to the 3 ' end.

[0297] In a control reaction with an RNA template but no dNTP added, a significantly lower number of RCPs are counted compared to in the positive reaction (with template and dNTP), indicating that gap filling by polymerization and triple helix formation is restricted, thus significantly reducing flap cleavage and therefore producing fewer ligation products ( Figure 11 A). The same trend is visible in the in situ reaction ( Example 5 - Use of Phi29 as a Reverse Transcriptase B and C).

[0298] Figure 13

[0299] Materials and Methods

[0300] Oligonucleotides

[0301] The oligonucleotide sequences are shown in Tables 8 and 9. Probes are provided as described in Example 1. A ligation reaction is carried out on a synthetic KRAS mRNA template. The padlock probe is designed such that after RNA hybridization, the probe circularizes, thereby forming a nick between the terminal arms. To facilitate the assessment of the size of the rolling circle product (RCP), a reporter sequence is embedded in the sequence of the ligation probe arm (backbone). Complementary decorations are used for RCP staining by hybridizing with the reporter sequence. For real-time RCA assessment, the amplified DNA is stained with SybrGold dye.

[0302] Real-time RCA. To evaluate the effect of RNA base pairs on the reverse transcription performance of Phi29 polymerase, 20 nM padlock probe was mixed with 10 nM RNA template supplemented with 4 U RNase inhibitor (DNA Gdansk), 3.75 U of PBCV-1 DNA ligase (SplintR, M0375S, NEB) in a final volume of 15 μL. The reaction was stirred at 37 °C for 30 min. After ligation, 2 μL of the ligation volume (loop) was mixed into an 18 μL RCA reaction mixture containing 1x Phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 mg / mL BSA (NEB), and 1×SybrGold (S11194, Invitrogen) to a final concentration of 2 nM loop. To ensure simultaneous initiation of RCA in all samples, the loops were placed in the tube caps and spun into the pre-disposed master mixture using a tabletop centrifuge. RCA was initiated immediately and SybrGold incorporation was detected using an Mx3005P qPCR system (Agilent Genomics) at 37 °C for 60 min, followed by Phi29 inactivation at 65 °C for 2 min.

[0303] To investigate whether the RCA efficiency of RNA-rich loops can be stimulated by adding reverse transcriptase, 100 U of RNaseH(-) TranscriptME reverse transcriptase (DNA Gdansk) was added to the RCA reaction mixture.

[0304] Sequencing of the RCA products generated from RNA-containing loops

[0305] Monomerization

[0306] To sequence the bases incorporated within the RCA products corresponding to the RNA bases within the circular template, the RCA products are first monomerized by restriction endonuclease digestion. First, the RCA products from the real-time RCA measurements as described above are diluted to a concentration of 100 pM in PBS-Tween 0.05%. Next, the RCA products are digested with AluI restriction endonuclease during a 10 min incubation at 37 °C in a reaction mixture containing 1x Phi29 DNA polymerase buffer, 2 mg / mL BSA, 100 nM restriction oligonucleotide (AluI KRAS RO - Table 9), 120 mU / μL AluI (NEB), and the RCA products at a final concentration of 10 pM and subsequently heat inactivated at 65 °C for 2 minutes. After complete digestion of 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 h RCA of an 80 base loop generates approximately 1000-fold amplification). The RCA monomers are diluted to 100 pM in PBS-Tween 0.05%.

[0307] Sequencing of the library preparation of RCA monomers

[0308] During the PCR reaction, RCA monomers were first labeled with Illumina adapter sequences. The PCR reaction contained 1x Taq DNA polymerase buffer (NEB), 1.5 mM MgCl2 (NEB), 250 μM dNTP, 1x SybrGold, 25 mU / μL Taq DNA polymerase (NEB), 0.5 μM forward primer PE1 (Table 9), 0.5 μM reverse primer PE2 (Table 9), and a final concentration of 10 pM RCA monomers. The PCR reaction started with denaturation at 95 °C for 5 min and was cycled 20 times between 95 °C for 15 s, 55 °C for 30 s, and 70 °C for 20 s. The reaction was monitored in a qPCR instrument and stopped before amplification reached saturation. After the first PCR step (extension step), 1 μl of the PCR product was incorporated into a mixture containing 1x Phusion HF buffer (Thermo Scientific), 0.2 mM d(A,T,G,C)TP (Thermo Scientific), 1% DMSO, 250 nM indexed PCR primers (Table 11), each sample being labeled with a unique combination of one of 7 different forward primers and one of 3 different reverse primers), and programmed for an initial 2 min at 95 °C, 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 mixture containing 1x Phusion HF buffer (Thermo Scientific), 0.2 mM d(A,T,G,C)TP (Thermo Scientific), 1% DMSO, 500 nM P5 and P7 primers, and programmed for 2 min at 95 °C, and 15 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were pooled and purified using the QIAquick PCR Purification Kit, and sequenced on the 550 System (Illumina) by 500hi-Output KT v2 (75CYS). Reads containing the correct primer sequences at the expected positions were extracted and analyzed with WebLogo.

[0309] Morphological assessment of RCP size and intensity

[0310] To measure the RCA product size and intensity, the RCA products from the real-time RCA reaction were diluted to a final concentration of 20 pM and labeled with a 5 nM final concentration of the decorating probe under standard hybridization conditions. 10 μL of the fluorescently labeled RCA products were applied onto Superfrost glass slides (Thermo Fisher), spread by a 20 x 20 mm coverslip (Menzel) and allowed to electrostatically bind to the positively charged surface during a 15-minute incubation. The coverslip was removed, the slides were briefly washed in PBS, fixed in the fixation medium, and imaged at 20x magnification in the Cy3 channel on a Zeiss Axioplan fluorescence microscope. The images were exported as raw black and white (BW) pictures and processed using Cell Profiler software. Briefly, each image was pre-processed using an automatic top-hat filter. Objects were identified using a manually adjusted threshold and separated based on object intensity. The average fluorescence intensity and object size were recorded, exported as a csv file and processed in R!Studio.

[0311] Results

[0312] Phi29 DNA polymerase accepts chimeric loops as rolling circle amplification (RCA) templates

[0313] We have observed that circular chimeric padlock probes containing 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 ( Figure 13 ).

[0314] The RCA reaction was monitored in real-time by SybrGold incorporation. Additionally, the RCA products were digitally counted and their size and intensity (i.e., morphology) were evaluated.

[0315] We observed that PBCV-1 readily seals pure DNA probes and chimeric 3 ' -(rN) / 5 ' (N) probe nicks, and Phi29 polymerase accepts pure DNA- and RNA-containing loops as templates for RCA ( Figure 13 A). A higher number of RCA products from the 3 ' RNA probe is likely due to increased ligation efficiency of the chimeric probe, as detailed in Example 1. When no template was added during the ligation reaction, the probes were not ligated and could not be amplified ( Figure 13 A, target -).

[0316] Next, we aimed to study the RCA efficiency of chimeric DNA / RNA loops in the absence of ligation reaction bias. To this end, we introduced RNA substitutions in the loop backbone that do not participate in the ligation reaction. Thus, all probes contained the same target-complementary DNA probe arm sequences (which contribute to the formation of ligation substrates) and were ligated to the same RNA targets. Then, we studied how increasing numbers of RNA substitutions affected the RCA reaction efficiency by counting the RCA products ( Figure 13 B) and monitoring the amplification reaction in real time ( Figure 13 C). Padlock probes with sequences SEQ ID NO:119, 120, 124, 131, 110, 111, and 112 as DNA probes and chimeric probes with 0, 1, 2, 3, 4, 5, and 7 consecutive ribonucleotides were used.

[0317] When a single RNA was substituted in the probe (loop) backbone, we did not observe an effect on the RCA efficiency ( Figure 32 B and 13C, Figure 13 A and 32B). When the loop was substituted with 2 consecutive RNA nucleotides, a strong inhibition of RCA (about 90%) was observed ( Figure 32 B and 13C, Figure 13 A and 32B). For loops with more than 2 RNA substitutions, no amplification was detected ( Figure 32 E, Figure 13 C). When the rolling circle products (RCPs) were imaged using an epifluorescence microscope, the average size and intensity of the RCPs of the loop with 2 consecutive RNA substitutions were reduced ( Figure 19 D). More than two consecutive RNA substitutions (3 - 7) led to complete inhibition of RCA and no RCPs were detected in these samples.

[0318] We also investigated whether the RCA of RNA-rich loops could be restored by supplying M-MuLV reverse transcriptase during the RCA reaction. However, under the reaction conditions used, the 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 panel of the curve) compared to the RCA reaction without additional reverse transcriptase ( Figure 18 ).

[0319] Phi 29 DNA polymerase preferentially reverse transcribes RNA pyrimidines during RCA

[0320] In previous experiments, DNA bases in the loop backbone were increasingly replaced by RNA bases. Strong amplification inhibition was observed for loops with rGrA and rGrArC replacements. To study whether the RCA efficiency of RNA-containing loops is sequence-dependent, we used loops with single rU / rA / rC / rG RNA bases as well as dinucleotide and trinucleotide-long homologous nucleotide segments to monitor the RCA rate in real time (Table 9). We observed efficient RCA for all single RNA replacements ( Figure 18 A). For dinucleotide RNA replacements, we observed the highest RCA rate for the rCrC loop, followed by the rUrU loop, while the rArA and rGrG loops were significantly inhibited. For trinucleotide RNA loops, only the rCrCrC loop produced detectable RCA, but at a significantly slower rate than the loop containing rCrC ( Figures 24 - 25 A). Many ribonucleotide probes SEQ ID NO:108 - 112, 124 - 127, 131 - 134, 137, and 139 contain other heteronucleotide ribonucleotide sequences. Experiments were repeated using probes SEQ ID NO:267 - 281 (Table 13) without additional ribonucleotide segments and similar results were observed ( Figure 18 ).

[0321] To study whether RCA of longer mixed RNA / DNA segments could be restored, we inserted 1 and 2 DNA bases into segments of 3 and 6 RNA bases, respectively ( Figure 18 B). Loops containing the rGArCGrU sequence in the backbone were amplified, while no RCA was detected for loops with 6 inserted RNA replacements, or loops with 5 and 7 consecutive RNA bases ( Figure 24 B).

[0322] Manganese ions increase the RNA-dependent RCA activity of phi29 DNA polymerase

[0323] Because some DNA-dependent DNA polymerases are able to reverse transcribe RNA in the presence of Mn 2+ , we compared the phi29 DNA polymerase RCA rate in the presence of Mg 2+ and Mn 2+ . Using Mn 2+ as a cofactor, phi29 DNA polymerase efficiently amplified single nucleotide, dinucleotide, and trinucleotide rU and rA segments in addition to rC ( Figure 24 ). Interestingly, the amplification rates of the rCrC, rUrU, and rArA loops were higher compared to loops with single RNA replacements, and this was also the case for rCrC with Mg 2+ as a cofactor ( Figure 25A, B). To study whether RCA could be restored if multiple RNA bases were mixed with DNA bases, chimeric constructs were amplified using Mg 2+ and Mn 2+ ( Figure 25 ). Based on our observations, phi29 DNA polymerase was able to participate in efficient Mn 2+ -dependent RCA ( Figure 25 , Table 12) when RNA bases were inserted into DNA. Interestingly, circular chimeric substrates with up to 8 RNA bases were efficiently amplified when the substitutions were organized in a uniformly dispersed pattern ( Figure 20 ).

[0324] Sequencing of the rolling circle products demonstrated the ability of Phi29 DNA polymerase to reverse transcribe RNA

[0325] Since the amplification rate of Phi29 polymerase was inversely proportional to the number of RNA bases in the substrate, we hypothesized that the enzyme might skip over RNA positions during RCA, introducing single- or dinucleotide deletions in the amplification products. To test this hypothesis, as described above, rings containing single and double RNA substitutions (rAr / Ur / G / rC / rArA / rUrU / rGrG / rCrC, Table 9) were amplified and the RCA rate was monitored in real time. After amplification, sequencing libraries were prepared from the different amplification products and then sequenced using the Illumina 550 system. Full-length sequencing reads were extracted from the dataset, aligned, and the base frequencies at each position in the padlock probe backbone were calculated ( Figure 20 ).

[0326] Based on our observations, Phi29 DNA polymerase incorporated the expected DNA nucleotides in amplified RCPs where the template sequence was RNA. For rings without RNA substitutions, >99% of the sequenced monomers showed correctly incorporated bases at the R1 padlock probe region (referred to here as accuracy), which was Figure 30Highlighted in the middle (99.68%, RT accuracy at position R1 of the DNA padlock probe). When the R1 position was replaced by rA, rC, rG or rU, the RT accuracies were 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 positions, rG stood out with a higher replication error. Interestingly, this higher incorporation error was observed not only for the R1 position, but also for all subsequent cytosine bases in the padlock probe backbone. When both the R1 and R2 positions were replaced by rArA, rCrC, rGrG and rUrU, the RT accuracies of the R1 / R2 sites were 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 a higher error rate for non-RNA cytosines in the entire probe backbone sequence.

[0327] 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 ( Figure 31 , Figure 33 , Figure 31 ). Although rA, rC and rU were copied with the same accuracy as DNA (as measured by sequencing), rG stood out with a higher replication error. Interestingly, a higher incorporation error was observed not only for the R1 position, but also for all guanine bases in the padlock probe backbone (visible as high error rate peaks in both Figure 33 and Figure 30 ), and a higher thymine frequency was observed for the rG padlock probe signature map in Figure 3 . When both the R1 and R2 positions were replaced by rArA, rCrC, rGrG and rUrU, the error rates of 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 a higher error rate for non-RNA guanines in the entire probe backbone sequence ( Example 6 - In Situ Detection of mRNA Using Chimeric Probes C).

[0328] We demonstrated the limited reverse transcription activity of Phi29 DNA polymerase. We confirmed that a single RNA substituent in the circular template had no effect on RCA efficiency. However, we have found that when more consecutive RNA bases are substituted in the circular template sequence, the amplification is inhibited. 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 3 RNA spacer loops with DNA bases led to a partial recovery of RCA efficiency, indicating that RCA of RNA-containing loops is limited by single RNA base substitutions or very short segments of consecutive RNA bases. Attempts to improve the RCA efficiency of loops containing longer segments 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 binding of the circular substrate to Phi29 DNA polymerase.

[0329] Our data clearly show that the mechanism by which the polymerase copies RNA-containing loops is reverse transcription, as we found that the matching DNA bases were incorporated into the RCA products at high frequencies (rA, rU, and rC were >99%, rG was approximately 96%). The overall incorporation accuracy of RNA substituents was indistinguishable from that of pure DNA substituents.

[0330] Figure 21

[0331] Materials and Methods

[0332] BjHtert and MEF cells were cultured in growth medium consisting of Dulbecco's Modified Eagle Medium (DMEM; Invitrogen), 10% fetal bovine serum (Sigma), and 1% penicillin-streptomycin mixture (PEST; Gibco). Both cell lines were grown at 37 °C in a humidified cell culture incubator in the presence of 5% CO2. Before the experiment, cells were removed from the culture flasks using 0.25% trypsin-EDTA solution (T4049 Sigma) and cultured overnight in a 150-mm cell culture dish with 5 submerged microscope slides. The slides with attached cells were washed twice with PBS and fixed for 15 min on ice in freshly prepared diethyl pyrocarbonate (DEPC)-treated PBS containing 3.4% formaldehyde. Thereafter, the slides were washed twice with DEPC-PBS, dehydrated in an ethanol gradient (70%, 85%, and 99%; 3 min each), air-dried, and stored at 80 °C. On the day of the experiment, the cells were thawed, dried, and separated for each test condition by covering with a secure seal chamber (Invitrogen) of 8 mm diameter and 50 μL volume. The cells were rehydrated with DEPC-TBS buffer. After each incubation step, there were two DEPC-PBS-T washes ((DEPC)-treated PBS containing 0.05% Tween 20 as surfactant). All incubations were carried out in a humid chamber to avoid evaporation of the reaction mixture.

[0333] Probes for the two ACTB transcripts (Table 4) were combined and pre-hybridized at 37 °C for 2 h in a 50 μL reaction volume in hybridization buffer (475 mM Tris-HCl at pH 8; 0.95 mM EDTA as shown in 4, 760 mM NaCl, 0.8 U / μL RNase inhibitor (DNA Gdańsk)) at a final concentration of 0.1 μM (Pool 1: non-chimeric PLP, Pool 2: chimeric PLP, Pool 3: iLock, Pool 4: chimeric iLock). Unhybridized probes were removed by stringent washing twice with pre-warmed (37 °C) TBS-Tween buffer. Ligation reactions were carried out 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 carried out by adding Taq DNA polymerase at a final concentration of 0.1 U / μL simultaneously. The slides were incubated at 37 °C for 2 h and washed twice with DEPC-PBS-T.

[0334] The rolling circle amplification reaction was carried out at 37 °C for 6 hours by adding 1 U / μL phi29 DNA polymerase (Monserate), 1x phi29 DNA polymerase buffer, 0.25 mM dNTP (Thermo Scientific), 0.2 μg / μL BSA (NEB), 5% glycerol and DEPC-ddH2O in a final reaction volume of 50 μL and washed twice with DEPC-PBS-T.

[0335] Finally, the decorated oligonucleotide was hybridized with the RCA product at a final concentration of 0.1 μM 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!.

[0336] Results

[0337] Although the probes were combined, only the expected signals were observed in the cells ( Figure 21 A). In addition, the chimeric padlock probes worked more effectively (produced more detectable RCA products) when compared to non-chimeric padlock probes. The iLock probes produced significantly fewer signals when compared to conventional padlock probes, indicating that the protocol needs to be further optimized to ensure efficient probe activation and in situ RNA detection. However, data analysis revealed that the expected signals were also observed for both chimeric and non-chimeric iLock probes, and the signals for the chimeric iLock probes were also higher ( Example 7 - Detection of miR21 on Solid Supports Using DNA PLP, Chimeric PLP, and DNA and Chimeric iLock Probes B).

[0338] Figure 22 Example 8 - In Situ Multiplex Gene Expression Profiling, Cell-Type Analysis, and In Situ Sequencing in Mouse Brain Tissue Sections Using Chimeric Padlock Probes

[0339] In this example, miR21 was immobilized on the slide surface and detected in situ. miR21 was prepared by separating the 5'-biotin moiety from the target sequence with a 16x rU linker. miR21 was detected with conventional and chimeric padlock probes and non-chimeric and chimeric iLock probes. The target sequences and probe sequences are shown in Table 5.

[0340] Materials and Methods

[0341] Place a safety-sealed chamber (Invitrogen) with a diameter of 8 mm and a volume of 50 μl on a neutrally biotin-coated microscope slide (PolyAn). A total of six safety-sealed silicone chambers were used. Dilute the miR21 target (miR21_BIO) to a final concentration of 50 nM in 1x labeling solution (2x SSC, 20% formamide) incubated at room temperature and gently shake for 1 hour. In one case, the miR21 target was intentionally omitted (negative control). After fixing the miR21, wash the chamber 3 times with PBS-Tween 20 (0.05%). Keep the chamber in which no ligation or activation occurred (coating control) in PBS until the end of the experiment.

[0342] Hybridize the padlock probe, iLock probe, and "coating control" probe (anti-miR21_FAM) to the immobilized target at a final concentration of 10 pM (padlock probe and iLock probe) or 50 nM (for the anti-miR21_FAM probe). Hybridize the probes 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 shake at room temperature for 3 hours. Then wash the chamber 2 times with PBS-Tween20 (0.05%).

[0343] Perform the ligation reaction by adding 0.5 U / μL SplintR ligase (NEB), 1x SplintR buffer in DEPC-ddH2O. Perform iLock ligation and activation (for iLock probes) by adding Taq DNA polymerase at a final concentration of 0.1 U / μL simultaneously. Incubate the slide at 37 °C for 1 hour and wash twice with DEPC-PBS-T.

[0344] Perform the rolling circle amplification reaction at room temperature for 3 hours with a final reaction volume of 50 μL by adding 0.5 U / μL phi29 DNA polymerase (Monserate), 1x phi29 DNA polymerase buffer, 0.125 mM dNTP (Thermo Scientific), 0.2 μg / μL BSA (NEB), 5% glycerol, and DEPC-ddH2O and wash twice with DEPC-PBS-T.

[0345] 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 h. Cells were washed twice with DEPC-PBS-T, dehydrated in 99% ethanol for 3 min, and the coverslips were mounted with Slow-Fade medium (Thermo Scientific). Signals in the cells were quantified using CellProfiler software.

[0346] Results

[0347] Our data showed that when miR21 was not fixed, the biotinylated miRNA targets were efficiently immobilized on neutrally biotin-coated microscope slides because no fluorescence was detected from the labeled complementary probes. The detection results are shown in and Cell-Type Analysis and In Situ Sequencing Panel B. Conventional padlock probes generated approximately 7800 rolling circle amplification products (RCPs) / field of view (fov), while approximately 36000 RCPs / fov were quantified when using chimeric padlock probes. Consistent with the example of detecting ACTB mRNA in BjhTERT and MEF cultured cells, the iLock probes generated fewer signals compared to padlock probes. The chimeric iLock probes generated approximately 3000 RCPs / fov, while the unmodified iLock probes generated only approximately 195 RCPs / fov.

[0348] Figure 23 ​

[0349] Materials and Methods

[0350] Immediately following surgical resection and without any fixation, P30 mouse brains were embedded in OCT medium and frozen directly on dry ice, and then stored at -80 °C until use. Then 10-μm sections were cut with a cryostat and the sections were collected on Superfrost glass slides. The sections were then briefly fixed in 3.7% PFA in DEPC-treated PBS at room temperature for 5 min. After that, the sections were washed once in 0.05% DEPC-PBS Tween and permeabilized with 0.1 M HCl at room temperature for 5 min. After permeabilization, the slides were washed twice in DEPC-PBS and dehydrated through a series of 70%, 85%, and 100% ethanol for 2 min each. A safety seal chamber was mounted on the slide covering the tissue section, and the tissue was hydrated by briefly washing with PBS-T (DEPC-PBS containing 0.05% Tween). To target mRNA with chimeric padlock probes (PLPs), after a brief rehydration wash, the sections were immersed in a chimeric PLP hybridization mixture containing 2x SSC buffer, 20% formamide, 0.05 M KCl, 0.2 mg / mL BSA, 1 U / μL RNase inhibitor, and 50 nM chimeric PLP. Hybridization was carried out overnight at 45 °C. After that, the sections were washed in pre-warmed buffer (2x SSC, 20% formamide) at 37 °C for 15 min. Finally, the sections were washed twice in PBS-T. Then a ligation reaction mixture was added to the sections, the ligation reaction mixture containing 1x SplintR ligase buffer, 0.2 mg / mL BSA, 0.8 U / μL RNase inhibitor, and 0.25 U / μL SplintR ligase. The ligation reaction was incubated at 37 °C for 60 min. The sections 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 dNTP, 0.2 mg / mL BSA, 1 U / μL phi29 polymerase, 5% glycerol, and 50 nM RCA primer. RCA was carried out at 37 °C for 3 h. Subsequently, the sections were washed twice in PBS-T and a detection probe (serving as an anchor probe in the in situ sequencing reaction) was in situ hybridized to the RCA products in 2x SSC and 20% formamide hybridization buffer at room temperature for 30 min.

[0351] For in situ sequencing, as previously described in Ke et al. (2013, Nature methods), sections were immersed in a ligation sequencing mixture containing 1x T4 ligation buffer, 1 mM ATP, 0.2 mg / mL BSA, 0.1 U / μL T4 DNA ligase, and 100 nM of each sequencing library base 1 (for sequencing the first barcode position, sequencing library base 2 for sequencing the 2nd barcode position, etc.). The sequencing reaction was incubated at room temperature for 1 h. The sections were then washed 3 times in PBS-T and the nuclei were stained with DAPI, washed three more times, subjected to a short ethanol series as described above, and then the tissue was fixed in Slowfade fixation medium. The tissue sections were then imaged in a fluorescence microscope with a 20x objective. To sequence the 2nd base, the sections were first washed in ethanol to remove the fixation medium and then washed 2 times in 100% formamide to strip the anchor probe and the ligated sequencing probe. The sections were washed 3 times in PBS-T and then the ligation sequencing mixture for the second base (same composition as above) was added to the sections and the procedure was repeated for the 3rd and 4th positions. 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).

[0352] Results

[0353] Due to the low efficiency of cDNA synthesis, multiplexed in situ gene expression profiling using cDNA synthesis and subsequent targeting of cDNA by padlock probes (PLPs) is typically limited to highly expressed genes. To date, direct targeting of RNA with PLPs has been difficult due to low probe ligation efficiency of the enzyme to RNA and insufficient specificity resulting in false positive signals. In this experiment, we demonstrated highly efficient ligation of chimeric PLPs to RNA ( ​)。We applied chimeric PLPs targeting 18 different genes, with 5 probes for each gene (90 probes in total) (Table 10) on mouse brain tissue sections. The probes were barcoded with sequencing barcodes that could be decoded by in situ sequencing later. First, the probes were hybridized to RNA and then ligated using the SplintR ligase after washing. The use of T4 RNA ligase 2 can further improve specificity as we have demonstrated an increase in specificity and efficiency using T4 RNA ligase 2 (see previous examples). The ligated probes were amplified by 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 patterns obtained using the direct RNA method with chimeric PLPs were very comparable to those 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 the potential for highly multiplexed RNA analysis in tissue sections in combination with in situ sequencing readouts.

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Claims

1. A method for detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, the method comprising: a) contacting the sample with a padlock probe and hybridizing the probe to the target nucleic acid molecule; b) performing a ligation reaction on the sample using a DNA / RNA ligase to ligate and thereby circularize any probe that has hybridized to the target nucleic acid molecule; c) amplifying the ligated circularized probe from step (b) by rolling circle amplification with a DNA polymerase; and d) detecting the amplification product from step (c) to thereby detect the target nucleic acid sequence; wherein the padlock probe is provided in one or more parts, each part having at least one target-specific binding site that is complementary to a homologous probe binding site at or adjacent to the target nucleic acid sequence and hybridizes to the target nucleic acid molecule such that, optionally after the step of cleaving the hybridized probe and / or extending its 3'-end using the target nucleic acid molecule as a template, the target nucleic acid molecule is used as a ligation template to juxtapose the ligatable ends of the probe or probe parts to ligate to each other, thereby creating a ligation site at or adjacent to the target nucleic acid sequence; and wherein optionally after the cleavage and / or extension step, the probe contains at least one ribonucleotide at or near the ligation site, and the ligated circularized probe consists mainly of DNA and contains no more than 4 consecutive ribonucleotides.

Images