Method for detecting a target nucleic acid sequence

Abstract

The present invention provides a method for detecting target nucleic acid molecules in a sample using padlock probes and rolling circle amplification. The method herein provides a new method for removing free padlock probes that have failed to hybridize or ligate to a target, and involves the use of a "silencing" oligonucleotide to "block" such unbound or unligated padlock probes.

Description

[Technical Field]

[0001] The present disclosure and invention relate to the field of nucleic acid detection. In particular, the present disclosure and invention relate to a method for detecting nucleic acid target molecules using padlock probes and rolling circle amplification (RCA). The method herein provides a new method for removing free padlock probes that have failed to hybridize or ligate to a target, and involves the use of oligonucleotides (referred to herein as silencing oligonucleotides) to "block" such unbound or unligated padlock probes. Also provided are silencing oligonucleotides and kits for use in such methods. [Background technology]

[0002] Detection of target nucleic acid molecules has applications in many different fields, including, inter alia, clinical disease diagnosis, prognosis and / or treatment, as well as research and biosecurity. Additionally, nucleic acids are frequently used as tags or labels in reporter systems for detecting other analytes, where the nucleic acid is detected as a surrogate or indicator of the analyte of interest.

[0003] Although target nucleic acid sequences can be easily detected using labeled hybridization probes, simple hybridization probes have a relatively high detection limit and cannot be readily used to distinguish between similar nucleic acid sequences.To improve sensitivity, target nucleic acid molecules containing target sequences are usually amplified to increase the amount of target sequences available for detection.RCA is commonly used for such amplification, and is usually used in conjunction with a padlock probe.

[0004] RCA uses a strand-displacing polymerase enzyme and requires a circular amplification template, which can be provided by a circularized padlock probe. Amplification of the circular template results in concatenated RCA products containing multiple copies of sequences complementary to those of the amplification template. Such concatemers typically form balls or "blobs," which are easily visualized and detectable, and RCA-based assays have been employed as reporter systems for the detection of nucleic acids, and indeed, more generally, for the detection of any target analyte.

[0005] Padlock probes are typically linear oligonucleotides with two separate target-complementary binding regions connected by an intervening "backbone" region. When a probe binds (hybridizes) to its target nucleic acid sequence, the ends of the probe can be ligated together to circularize the probe. The circularized padlock probe can then be used as a template for an RCA reaction, and the RCA product can be detected. This forms the basis of several detection assays currently in use. Padlock probes are highly specific because they require dual recognition, or two binding sites, for the target nucleic acid sequence. They also provide an additional layer of specificity, as only probes that are correctly base-paired at the ligation site are ligated to generate the molecular template that is detected. When a padlock probe hybridizes to a target nucleic acid sequence with its target-binding regions directly adjacent to each other, the ends of the padlock probe can be directly ligated to each other. Alternatively, the target binding regions of a padlock probe may hybridize to a target nucleic acid with a gap between them, and the gap may be filled either by hybridization of one or more gap oligonucleotides in the gap region or by polymerase-catalyzed extension at the hybridized 3' end of the probe. In this way, the hybridized ends of a padlock probe may be indirectly ligated to each other in that they each hybridize to an intervening "gap sequence." Such "gap-fill" padlock probes are also known as molecular inversion probes.

[0006] Generally speaking, in RCA-based methods using padlock probes, it is desirable to remove unbound padlock probes, i.e., padlock probes that have not hybridized to the target nucleic acid (i.e., free padlock probes) or have not been ligated before the RCA reaction is performed. In this way, mispriming by free padlock probes present in the reaction mixture (e.g., by hybridizing to the product of the RCA reaction) can be avoided. More specifically, free padlock probes that subsequently hybridize to the RCA product (RCP) generated in the RCA reaction can prime an extension reaction in which the RCP serves as a template, resulting in a double-stranded product to which a detection oligonucleotide for detecting the RCP cannot bind. Removing or "neutralizing" such free padlocks may improve the specificity and / or sensitivity of the method because they cannot participate in subsequent hybridization or priming reactions.

[0007] Current means for removing free padlocks include washing. However, this requires a solid phase and is therefore not applicable to homogeneous or so-called "in-solution" methods that do not use a solid phase. Free padlocks may also be removed by enzymatic digestion, particularly using an exonuclease enzyme that can digest nucleic acids with free or unligated ends but does not address circularized padlock probes that do not have free ends. However, the disadvantages of this are not only the expense of additional enzymes, but also the need for exonuclease inactivation to prevent digestion of other downstream assay / reaction components (e.g., including RCPs). Such inactivation is typically achieved by heating to 95°C. An alternative solution is needed to enable assays to be performed in solution in a single tube without additional enzymatic, heating, or washing steps. Summary of the Invention

[0008] The present inventors have devised an alternative solution to the problem of removing unwanted and unhybridized padlock probes in padlock probe and RCA-based assay methods.

[0009] This involves the use of oligonucleotides (herein referred to as "silencing oligonucleotides") that can hybridize to the ends of padlock probes and prevent them from participating in further reactions. In effect, the padlock probes become "closed" and functionally inactivated unless the correct template is present. In other words, the padlock probes become inactive. To ensure that the ends of the padlock probes cannot be ligated to each other in a ligation reaction in which the silencing oligonucleotide serves as the template, the padlock probe binding regions of the silencing oligonucleotides are separated by a gap, which keeps the ends of the padlock probes separated from each other when the padlock probes are hybridized to the silencing oligonucleotides.

[0010] Thus, in a first aspect, there is provided herein a method for detecting a target nucleic acid molecule in a sample, the method comprising: (i) contacting the sample with a padlock probe that contains target binding regions at its 5' and 3' ends that are complementary to probe binding sites in a target nucleic acid molecule; (ii) contacting the padlock probe with a silencing oligonucleotide comprising first and second silencing regions complementary to the target binding region at the end of the padlock probe, wherein the two silencing regions are separated by at least 5 nucleotides, said contacting occurring before, during, or after contacting the sample with the padlock probe; (iii) juxtaposing and hybridizing the target-specific binding regions of the padlock probe to the target nucleic acid molecule for direct or indirect ligation to each other; (iv) directly or indirectly ligating the 5' and 3' ends of the padlock probe to circularize the padlock probe; (v) after the silencing oligonucleotide contacts the padlock probe, performing an RCA reaction using the circularized padlock probe as an RCA template to generate an RCA product (RCP); (vi) detecting the RCP during or after the RCA reaction to detect the target nucleic acid molecule.

[0011] Silencing oligonucleotides can be thought of as blocking oligonucleotides relative to padlock probes. Furthermore, because silencing oligonucleotides perform the equivalent function of washing away unhybridized or unligated padlocks, silencing oligonucleotides may also be referred to as "washing oligonucleotides" (and similarly, the silencing region of an oligonucleotide may alternatively be referred to as a "washing region").

[0012] A feature of this method is that the RCA reaction does not occur, i.e., is not initiated, until after the step of contacting the silencing oligonucleotide with the padlock probe. In other words, the RCA begins after the silencing oligonucleotide is present in the sample or reaction mixture (and is available to bind to any free padlock probe). More specifically, the RCA reaction occurs (i.e., is initiated) after contact with the silencing oligonucleotide and after ligation of the target-hybridized padlock probe. More specifically, the RCA reaction does not begin until after the contact or after the contact and ligation, which may be shortly thereafter.

[0013] An RCA reaction is typically initiated by adding one or more reagents for RCA (referred to as "RCA reagents"). This is typically a polymerase enzyme and nucleotides (specifically dNTPs) for RCA, although primers for RCA may also be added in some cases. One or more of the RCA reagents may be added in advance only if all of the RCA reagents are not already present. In certain embodiments, the RCA reaction is initiated by adding at least a polymerase enzyme.

[0014] This method is particularly suitable for a homogeneous solution format. Thus, in some embodiments, the method is carried out in solution, i.e., in the absence of a solid phase, at least up to step (v). In other words, in some embodiments, the padlock probe contacting step, the silencing oligonucleotide contacting step, the padlock probe hybridization and ligation step, and the RCA step are carried out in solution or in the absence of a solid phase.

[0015] In an advantageous embodiment, the method is carried out in a single reaction vessel (eg, a single tube).

[0016] As discussed in more detail below, the target nucleic acid molecule may be the analyte, or it may be a nucleic acid molecule used or generated as a surrogate, or reporter or indicator, of the analyte to be detected.

[0017] In some embodiments, the silencing oligonucleotide is pre-hybridized to, pre-mixed with, or added to the sample together with the padlock probe, while in other embodiments, the silencing oligonucleotide is contacted with the sample after the padlock probe has hybridized to or been ligated to the target nucleic acid molecule.

[0018] In certain embodiments, the silencing oligonucleotide comprises a third silencing region located between the first and second silencing regions, such that the silencing oligonucleotide binds to the padlock probe at three sites.

[0019] In another aspect, there is also provided herein a kit for detecting a target nucleic acid molecule in a sample, the kit comprising: (i) a padlock probe comprising target binding regions at its 5' and 3' ends that are complementary to probe binding sites in a target nucleic acid molecule; (ii) a silencing oligonucleotide comprising first and second silencing regions complementary to the target binding region at the end of the padlock probe, wherein the two silencing regions are separated from each other by at least 5 nucleotides.

[0020] As described above, the silencing oligonucleotide may optionally contain a third silencing region. Thus, the stretch of nucleotides (i.e., the nucleotide sequence) separating the first and second silencing regions may or may not hybridize to the back piece of the padlock. The back piece is the portion of the padlock that is between the target binding regions.

[0021] In this aspect, the padlock probe and the silencing oligonucleotide may be as defined above.

[0022] In yet another aspect, provided herein is a silencing oligonucleotide for blocking a target binding region of a padlock probe, wherein the silencing oligonucleotide comprises first and second silencing regions complementary to the target binding regions at the ends of the padlock probe, the two silencing regions being separated from each other by at least 5 nucleotides, and the silencing oligonucleotide further comprises a third silencing region located between the first and second silencing regions and complementary to a silencing oligonucleotide binding site in the padlock probe located between the target binding regions in the padlock probe. [Brief explanation of the drawings]

[0023] [Figure 1] 1 shows a reaction scheme for detecting a target nucleic acid molecule using a padlock probe. The target-binding regions at the 5' and 3' ends of the probe hybridize directly adjacent to each other on the target nucleic acid molecule. The two ends are ligated together using a ligase enzyme to form a circular template that is amplified by RCA using Phi29 polymerase to generate a long, linear, concatemeric RCP. The RCP is detected using a labeled detection oligonucleotide. [Figure 2] Figure 1 shows hybridization of a padlock probe to a silencing oligonucleotide. (A) A silencing oligonucleotide with two silencing regions, each complementary to an end of the padlock probe. The two silencing regions are separated by a sequence that is not complementary to the padlock probe, resulting in a gap between the hybridized ends of the probe. (B) and (C) Alternative configurations for hybridization of a padlock probe to a silencing oligonucleotide with a third silencing region that hybridizes to the middle portion ("backbone" or "backpiece") of the padlock probe, located between the two end regions. [Figure 3]This figure shows the results of an experiment using the model system described in Example 1. Synthetic templates containing nine different target sequences were added to a set of tubes (referred to as "positive samples"). A control set of tubes (referred to as "negative samples") contained no synthetic templates but instead synthetic non-target template molecules (referred to as "incorrect templates"). A ligation mix containing nine padlock probes targeting the nine target sequences, nine cognate silencing oligonucleotides (one for each padlock probe), and ligase was added to the tubes and incubated. An RCA mix containing reagents and detection beads for RCA was added, and RCA was performed. The top panel shows eight replicate assays of the same positive sample, and the bottom panel shows eight replicate assays of the same negative sample. [Figure 4] (A) Results of an experiment using the model system of Example 1 comparing the correct target molecule (synthetic template) with an incorrect template molecule; (B) Titration of silencing oligonucleotides in the model system (5 nM padlock probe, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 nM silencing oligonucleotide). [Figure 5] Results of testing of clinical samples are shown. Samples in tubes 1-20 were confirmed COVID-19 positive by PCR, and samples in tubes 21-40 were confirmed COVID-19 negative by PCR. (A) shows results in the absence of silencing oligonucleotides, and (B) shows results in the presence of silencing oligonucleotides. [Figure 6]Results from a model system are shown, demonstrating the improved sensitivity achieved with triple-binding silencing oligonucleotides. (A) Titration of template oligonucleotide in the model system without silencing oligonucleotides. (A) Template oligonucleotides at concentrations of 5 nM, 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, and 5 fM, as well as a negative control (Neg C), demonstrate binding of different detection oligonucleotides to either rolling circle products (RCPs) or free padlocks (which, in this experiment, are not inhibited by the silencing oligonucleotides from interacting with RCPs). The detection limit here is between 5 pM and 50 pM. (B) Titration of template oligonucleotide in the model system together with triple-binding silencing oligonucleotides. (B) shows the template oligonucleotide and negative control (Neg C) at concentrations of 5 nM, 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, and 5 fM, together with the silencing oligonucleotide at a 1:1 ratio relative to the padlock. Demonstrated here is the growth of an RCP without the interference of a free padlock, in which the silencing oligonucleotide binds to the growing padlock using one arm. If the padlock is able to bind with one of its arms, the RCP becomes double-stranded. Only complete binding of the padlock to the growing RCP is possible (because the padlock prefers binding to the complete target (18 + 18 nt (36 nt)) over the triple-binding silencing oligo. However, if the free padlock binds to the RCP using one arm, the silencing oligo then binds to the other arm, and phi29 cannot make the product double-stranded). This then allows for second-generation RCPs. The silencing oligonucleotide allows complete binding of the padlock to the template, which improves the sensitivity of the assay to a detection limit between 5 fM and 50 fM. [Figure 7]Data are presented from a model system comparing two silencing oligonucleotide designs and a negative control (Neg C) at different template oligonucleotide concentrations (5 nM, 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, and 5 fM) containing the silencing oligonucleotide at a 1:1 ratio relative to the padlock. The "double-binding" silencing oligonucleotide in (A) has two silencing regions (binding at two positions), compared to the "triple-binding oligonucleotide" in (B) with three silencing regions (binding at three positions). The results show that the silencing oligonucleotide with three silencing regions allows for a second round of RCA (on top of the first round of RCA). This only occurs when the silencing oligonucleotide binds to the padlock at three positions (B). The silencing oligonucleotide that does not bind to the padlock backpiece (A) does not allow for this lower sensitivity—it does not allow for a second round of RCA. Silencing oligonucleotides with three binding sites for the padlock allow complete binding of the padlock to the template, which improves the sensitivity of the assay to a detection limit between 50 fM and 5 fM, compared to a detection limit between 50 pM and 5 pM for those with two binding sites. [Figure 8] Two different designs of silencing oligonucleotides with three silencing regions (binding sites to padlock probes) are shown. (A) is in a "Danish" conformation (BD), and (B) is in an "omega" conformation (BO). The first and second silencing regions, which bind to the target binding regions at the ends of the padlock probe, are referred to as "arms," ​​and the third silencing region, which binds to the middle (backpiece) portion of the padlock probe, is referred to as Bp ("backpiece"). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present method provides an improved method for detecting a target nucleic acid molecule in a sample. More specifically, the method can be used to detect a specific nucleotide sequence in a target nucleic acid molecule, or in other words, a target nucleotide sequence (the term "nucleotide sequence" may be used interchangeably with "nucleic acid sequence").

[0025] The method is based on detecting a target nucleic acid using a padlock probe, which, upon binding to its target, is ligated and subjected to an RCA reaction. The product of the RCA reaction is detected to detect the target nucleic acid by detecting the padlock probe. The improvement relates to an improved method for removing or eliminating unhybridized and unligated free padlock probes. Free padlock probes are eliminated by blocking them using silencing oligonucleotides to render them inactive, so that they cannot participate in any nonspecific or unwanted reactions that interfere with the assay and / or generate nonspecific signals. In this way, the specificity and / or sensitivity of the assay method herein can be improved. Therefore, "removal" or "elimination" herein does not require physical removal in the sense of separation from the sample, but rather includes functional removal or elimination. When silenced or blocked by a silencing oligonucleotide, the padlock probe cannot be ligated or is substantially unavailable for ligation. Furthermore, once hybridized to a silencing oligonucleotide, it becomes unavailable or substantially unavailable for hybridization to any other nucleic acid molecule that may serve as a template for RCA, and therefore it is unable or substantially unable to prime an extension reaction against any other nucleic acid template that may be present or available in the sample.

[0026] As described in more detail below, in padlock probe-RCA-based detection methods, RCA products (RCPs) can typically and preferably be detected by hybridizing a detection oligonucleotide to the RCP, which is then detected in the RCP. To bind to the RCP, the detection oligonucleotide must remain in the single-stranded form generated by the RCA of the circularized padlock probe. Therefore, it is important to prevent the RCP from becoming double-stranded, which could result in unwanted extension reactions due to the RCP being used as an extension template. Thus, it is undesirable for free padlock probes that have not hybridized to target nucleic acid molecules or padlock probes that have not been ligated in a previous step to be available to hybridize to the RCP and prime an extension reaction thereon.

[0027] Thus, in one embodiment, the purpose of the silencing oligonucleotide is to effectively bind to, and thus sequester, any free, unbound or unligated padlock probes present in the reaction mixture (the mixture resulting from contacting a sample with a padlock probe and hybridizing or hybridizing and ligating the probes). Thus, in this embodiment, the silencing oligonucleotide acts to prevent, or reduce or inhibit, any free padlock probes present in the reaction mixture, i.e., padlock probes that are not hybridized or ligated to a target nucleic acid, from hybridizing to, or being able to hybridize to, an RCP.

[0028] Thus, the silencing oligonucleotide is contacted with the padlock probe before the RCA is performed (ie, before the RCA reaction is initiated).

[0029] However, while the binding of unreacted padlock probes to RCPs is generally undesirable in RCA reactions, in so-called super RCA (sRCA) reactions, padlock probes are used to bind to first-generation (or first-round) RCPs to perform a second-round RCA reaction using the first RCP as a ligation template for the padlock probe. This results in the generation of a second-generation RCA product (the so-called "second RCP") by RCA of the padlock ligated on the first RCP. We have found that the design of certain silencing oligonucleotides, i.e., those with three silencing regions, can promote or enable such a second-round RCA reaction (or, in other words, sRCA). This is described in more detail below.

[0030] In certain embodiments, the cognate pair of silencing oligonucleotide / padlock probe can be designed to minimize the possibility of displacement of the silencing oligonucleotide from its hybrid with the padlock probe by RCP once it is generated. Furthermore, by including a detection oligonucleotide in the RCA reaction mix, the detection oligonucleotide can hybridize to the RCP once it is generated, thereby minimizing the risk of such displacement. Furthermore, the silencing oligonucleotide / padlock probe hybrid may itself hybridize to the RCP via another portion of the padlock probe. Such a bound padlock cannot be amplified by RCA.

[0031] In other embodiments, the cognate silencing oligonucleotide / padlock probe pair can be designed to favor binding of the padlock probe to its target (or "template"), such that in the presence of the target sequence, the padlock probe binds to the target sequence rather than the silencing oligonucleotide. This is the case for sRCA reactions, where a second round of RCA is desired.

[0032] By "removing" or blocking unreacted padlock probes in this manner, the method finds itself particularly useful for homogeneous assays, i.e., assays carried out in solution without a solid phase, although nothing prevents its application in heterogeneous or solid phase-based methods.

[0033] As noted above, the use of padlock probes and RCA to detect target nucleic acids according to the methods herein is known in the art. The operation of such a detection method is shown schematically in FIG. 1. The padlock probe hybridizes to the target nucleic acid. As discussed in more detail below, depending on the configuration of the padlock probe, the target binding region of the padlock probe may be capable of hybridizing to (or, in other words, complementary to) a target sequence in a target molecule to be detected. Alternatively, the target binding region of the padlock probe may be capable of hybridizing to (or complementary to) a binding site in the target nucleic acid molecule that is adjacent to the target sequence. The latter option may be applicable, for example, when the padlock probe is a gap-filling padlock. In this case, the gap between the hybridized 5' and 3' ends of the padlock probe is filled by polymerase-catalyzed extension of the hybridized 3' end of the padlock using the target nucleic acid molecule as an extension template. The padlock probes contact target nucleic molecules in the sample and hybridize to the target molecules.

[0034] The term "contacting" is used broadly herein to include any means of contacting a sample, or more specifically, a target nucleic acid molecule. This may include, for example, adding a padlock probe to the sample and maintaining, e.g., incubating, the resulting reaction mixture under conditions that allow the probe to hybridize to the target nucleic acid molecule. Alternatively, the sample, or an aliquot or portion thereof, may be added to a padlock probe or a reaction mix that includes a padlock probe.

[0035] Upon hybridization of the padlock probe to the target nucleic acid, the ends of the padlock probe are juxtaposed for ligation to one another (directly, if the ends hybridize directly adjacent to one another, or indirectly, if the ends of the padlock probe hybridize with a gap between them). As described above and in more detail below, the gap may be filled by a gap oligonucleotide or by gap-filling extension. As shown in FIG. 1, the ends of the padlock probe hybridize directly adjacent to one another, so that they can be directly linked by ligation. The padlock probe is circularized by ligation to generate a circular template for the RCA reaction. The RCA reaction generates a long concatemeric product (RCP) containing multiple repeats of complementary copies of the circularized padlock probe linked in tandem. In other words, the RCP contains multiple monomers linked to one another, each representing a complementary copy of the circularized padlock probe. As shown in Figure 1, the padlock probe contains a detection sequence that is copied into each repeat of the monomer in the RCP, providing a binding site for the detection oligonucleotide. Hybridization of the detection oligonucleotides to multiple binding sites concentrates them in the RCP, allowing for highly sensitive detection. As described in more detail below, the detection oligonucleotides may be attached with a detectable moiety, e.g., a label, to enable detection. As shown in Figure 1, the label is a colored bead, but any detectable label may be used.

[0036] Any padlock probes that fail to hybridize to the target or fail to ligate may remain free in the sample, or more specifically, in the reaction mixture. To prevent such unbound or unligated padlock probes from hybridizing to or being available to hybridize to the generated RCP and from priming or being available to prime undesired extension reactions, silencing oligonucleotides are used to "capture" or "block" such probes before the RCA reaction is initiated. More specifically, the target-binding region of the padlock probe is blocked by binding to a silencing oligonucleotide, for example, as shown in Figure 2.

[0037] Most generally, the method is for detecting a target nucleic acid molecule. More specifically, the method is for detecting a target nucleic acid sequence in a target nucleic acid molecule. The term "detecting" is used broadly herein to include any means of determining the presence of a target nucleic acid molecule. In the method, the target nucleic acid is detected by detecting the presence or amount of the generated RCP, which may include simple detection of its presence or absence or any form of measurement of the RCP. Thus, the RCP product generated in step (v) can be detected as a "signal" of the target nucleic acid molecule. Therefore, detecting the RCP in step (vi) includes determining, measuring, evaluating, or assaying the presence or absence, amount, or location of the RCP in any manner. Because successful generation of the RCP depends on the presence of the target nucleic acid molecule (or more specifically, the presence of the target nucleic acid sequence therein), the presence of the RCP (i.e., confirmation of its presence or amount) is an indicator that indicates or identifies the presence of the target nucleic acid molecule.

[0038] Quantitative and qualitative determinations, measurements, or assessments are included, including semi-quantitative. Such determinations, measurements, or assessments may be relative, for example, when two or more different target nucleic acid sequences or target molecules are detected in a sample, or may be absolute. Thus, in certain embodiments, the method may be for quantifying or determining the amount of a target nucleic acid molecule or sequence present. The term "quantifying" when used in quantifying a target nucleic acid molecule(s) or sequence(s) in a sample may refer to absolute or relative quantification. Absolute quantification may be achieved by including one or more control nucleic acid molecules of known concentrations and / or by referencing the detected level of the target nucleic acid molecule or sequence with known control nucleic acid molecules or sequences (e.g., through the generation of a standard curve). Alternatively, relative quantification may be achieved by comparing the detected levels or amounts between two or more different target nucleic acid molecules or different target sequences to provide a relative quantification of each of the two or more different nucleic acid molecules or sequences (i.e., relative to each other). Thus, as described above, the ratio of target nucleic acid molecules or sequences present in a sample can be determined. Thus, for example, the copy number of target nucleic acid molecules, eg, chromosomes, can be compared.

[0039] A target nucleic acid molecule is any nucleic acid molecule to be detected, or in other words, a nucleic acid molecule that is the target of an assay. More specifically, the target may be a target sequence present in a nucleic acid molecule. In some embodiments, it may be desirable to detect two or more target sequences present in a target molecule. In other embodiments, two or more target molecules or two or more target sequences present in two or more target molecules may be detected. The method may be performed in a multiplex manner, for example, to detect two or more different target sequences in one or more target nucleic acid molecules and / or target sequences in two or more target molecules. In this way, to detect target sequences in two or more different target molecules, multiple first padlock probes may be used, each specific for a different target sequence, i.e., complementary to a binding site in the target sequence, or, for example, having target binding regions adjacent to different target sequences. In this regard, it will be understood that if the target binding sites are adjacent to the target sequence in a molecule, adjacent binding sites in different target molecules may be different for different target sequences to allow specific binding of the padlock probes. Thus, in such situations, the adjacent sequences targeted by the padlock probes are discriminatory between the different targets, and therefore may be considered part of the target sequence. Alternatively or additionally, different and distant target sequences within the same target molecule, for example, different sequences in different genes on a chromosome, may similarly be detected using multiple different padlock probes, each specific for a different target sequence.

[0040] In certain embodiments, the methods are useful for detecting whether multiple possible different variant sequences are present in a given target molecule, for example, whether a wild-type or mutant sequence is present, or whether multiple possible mutations, or different allelic variants, or polymorphisms, etc. are present. In such protocols, padlock probes may contain target binding regions designed to distinguish between different variants. Alternatively, gap-fill padlock probes may be used that hybridize to adjacent regions flanking all or multiple variants, and a gap-fill extension step is performed to generate complementary copies of the variant sequence(s) present. By detecting the complementary sequences of the variant sequences present in the RCP, the nature of the variant sequence can be determined or the variant can be detected. This may be, for example, by sequencing or by using detection oligonucleotides specific for the variant sequence.

[0041] Thus, the present method may be used to detect multiple target molecules or target sequences. As used herein, the term "multiple" means two or more, e.g., 3, 4, 5, 6, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 or more. In practice, thousands or tens of thousands of padlock probes may be used. The number of padlocks that can be used is not limited and can vary. This depends on the purpose of the method, the nature of the sample, the target nucleic acid sequence to be detected, the number of possible variants, and the like. Thus, for example, to detect wild-type and mutant variants, the number of different padlocks depends on the number of possible different variants, and may be, for example, 2-6, 2-5, 2-4, or 2-3 different padlocks. It will be understood that different aspects may be combined to increase the overall multiplexing of the assay. For example, in a given sample, the present method may be used to detect different variants of different target sequences.

[0042] The target nucleic acid molecule or sequence may be any molecule or sequence to be detected or identified. It may be DNA or RNA, or a modified variant thereof. Thus, the nucleic acid may be composed of ribonucleotides and / or deoxyribonucleotides, as well as synthetic nucleotides capable of participating in Watson-Crick or similar base pairing interactions. Thus, the nucleic acid may be or include, for example, bisulfite-treated DNA, LNA, PNA, or any other derivative containing a non-nucleotide backbone.

[0043] Typically, the target molecule or sequence is an analyte to be detected, e.g., a nucleic acid present in a sample, e.g., a cell or tissue sample, or any biological or clinical sample. It may be a viral nucleic acid. Thus, it may be a naturally occurring sequence, or a derivative, copy, or amplicon thereof. However, the method is not limited thereto, and the target molecule may instead be a reporter of the analyte in the assay. The reporter nucleic acid may be used or generated during the course of an assay for any analyte in the sample, e.g., a protein or other biological molecule, or a small molecule. Thus, the reporter nucleic acid may serve as a tag or label for a binding probe for the analyte, and may be detected, for example, in an immunoassay, e.g., in an immuno-PCR or immuno-RCA reaction, to detect the analyte. The reporter nucleic acid may be generated during the assay, e.g., by a ligation reaction in a proximity ligation assay, an extension reaction in a proximity extension assay, or by a cleavage reaction. Accordingly, such reporter target nucleic acids may be synthetic or artificial sequences.

[0044] In some embodiments, the target nucleic acid is a DNA molecule (natural or synthetic). The target nucleic acid molecule may be coding or non-coding DNA, for example, genomic DNA or a subfraction thereof, or may be derived from genomic DNA, for example, a copy or amplicon thereof, or it may be cDNA or a subfraction thereof, or an amplicon or copy thereof, etc.

[0045] In another embodiment, the target nucleic acid molecule is a target RNA molecule. It may be an RNA molecule or other nucleic acid molecule in a pool of RNA, for example, genomic nucleic acid, human or from any source, transcriptome, or any other nucleic acid (e.g., organelle nucleic acid, i.e., mitochondrial or plastid nucleic acid, or viral nucleic acid), naturally occurring or synthetic. Thus, the target RNA molecule may be or be derived from a coding (i.e., pre-mRNA or mRNA) or non-coding RNA sequence (e.g., tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA). In one embodiment, the target nucleic acid molecule is a microRNA (miRNA). In one embodiment, the target RNA molecule is 16S RNA, for example, where the 16S RNA is derived from and identifies a microorganism (e.g., a pathogenic microorganism) in the sample. Alternatively, the target RNA molecule may be genomic RNA, for example, ssRNA or dsRNA of a virus that has RNA as its genetic material. Notable such viruses include Ebola, HIV, SARS, SARS-CoV2, influenza, Hepatitis C, West Nile, polio, and measles. Thus, the target RNA molecule may be positive-sense RNA, negative-sense RNA, or double-stranded RNA derived from a viral genome, or positive-sense RNA derived from a retroviral RNA genome.

[0046] If the target molecule is an RNA molecule, the method may include the preliminary step of generating a cDNA copy of the target RNA molecule. The cDNA molecule is then contacted with a padlock probe in step (i).

[0047] Alternatively, the target RNA molecule may be directly contacted with the padlock probe, in other words, the first padlock probe may bind directly to the target RNA molecule.

[0048] For the detection of variant target sequences, as described above, padlock probes may be specific for a particular variant. However, as noted above, this is not a requirement, and in another embodiment, padlock probes capable of capturing any possible variant of a given target sequence are used. Thus, such padlock probes are not selective or specific for any particular variant. However, to ensure capture of the intended target sequence, they are designed to specifically bind to the target molecule at a site where all variants of the target sequence can be captured, i.e., at a site adjacent to the target sequence that is common among different variants. By way of example, a variant sequence may be a mutation or polymorphism at a particular position or locus in a gene. Thus, the target sequence may be a sequence that includes or constitutes the variant position or locus, and different target sequences may be distinguished by having different bases at that position or locus. Padlock probes may be designed to have binding sites that are complementary to adjacent sequences in the target molecule that are shared or conserved (i.e., common) among different variants.

[0049] Thus, in such embodiments, the padlock probe may be thought of as a common probe or as a generic probe for a group of target sequences or a group of variant target sequences. Thus, a padlock probe may have a target binding region that is complementary to a binding site (i.e., a flanking region) in a target molecule that is common to different target sequences (i.e., a common binding site that is flanked by different target sequences or different variants of a target sequence). Alternatively, a padlock probe may have a target binding region that is common or generic to different target sequences or different variants, or common to a group of target sequences or a group of variants. That is, a padlock probe may have a target binding region that can hybridize to a complementary binding site in a target molecule that is common to different target sequences or different variants of a target sequence.

[0050] In different embodiments, the padlock probe may be specific to a particular target nucleic acid sequence. Thus, for example, different padlock probes may be used, each specific to a different target sequence. This may be useful in diagnostic tests, such as NIPT, where different sequences may be detected, for example, to detect different chromosomes (and determine their copy number to detect trisomy), or in any situation where it is desired to detect one or more specific target sequences.

[0051] A padlock probe may alternatively be defined as a circularizable probe. The use of padlocks or circularizable probes, including in RCA reactions, is well known in the art. A circularizable probe comprises one or more linear oligonucleotides that can be ligated together to form a circle. Padlock probes are well known, widely used, and well reported and described in the literature. Thus, the principles of padlock probing are well understood, and the design and use of padlock probes are known and described in the art. A padlock probe is typically a linear circularizable oligonucleotide that hybridizes to a target nucleic acid sequence or molecule in a manner that juxtaposes the 5' and 3' ligatable ends of the probe for ligation to each other, either directly or indirectly with a gap between them, as described above. The probe is circularized by ligating the hybridized 5' and 3' ends of the probe. It is understood that for circularization (ligation) to occur, the ligatable 5' end of the padlock probe has a free 5' phosphate group.

[0052] To allow for juxtaposition of the ends of the padlock probe for ligation, the padlock probe is designed to have target binding sites at its 5' and 3' ends, i.e., the regions of complementarity that allow the padlock probe to bind to its target are located at the ends of the padlock probe.

[0053] To allow ligation, the 3' and 5' ends to be ligated (the "ligatable" 3' and 5' ends) hybridize to a target molecule or sequence, which acts as a template for ligation. Binding of the padlock probe juxtaposes the ends as described above. If complementary binding sites in the target molecule or sequence are located immediately adjacent (or contiguous) to one another, the ends of the padlock probe hybridize immediately adjacent to one another (i.e., without a gap) and can be directly ligated to one another. Thus, in this case, the ligatable end of the probe is constituted by the actual end of the probe. In the case of a gap-fill padlock probe, the target binding region at the end of the padlock probe does not hybridize to adjacent binding sites, but rather to non-adjacent (non-contiguous) binding sites in the target molecule. In such an arrangement, the 5' ligatable end of the probe is constituted by the actual 5' end of the probe. However, the ligatable 3' end of the probe is generated by extension of the hybridized 3' end of the probe using the target sequence as an extension template to fill the gap between the hybridized ends of the probe. The extension reaction juxtaposes the extended 3' ends of the probe for ligation. In this case, the ligatable 3' end of the probe is therefore the extended 3' end of the probe.

[0054] A padlock probe may be composed of two or more parts that are ligated together. For example, in the case of a two-part probe, where each part contains only one target-binding region and the other end of each part hybridizes to a common ligation template, this may involve the formation of an additional ligation template. In another embodiment, a two-part padlock may take the form of a "connector" or "backbone" oligonucleotide with two target-binding regions at or near its respective 5' and 3' ends (which hybridize to the target with a gap between them), and a gap oligonucleotide that hybridizes to the gap between the ends. The gap oligonucleotide can partially or completely fill the gap. In the case of a gap-fill padlock probe, the 3' end to be extended may be the hybridized 3' end of the gap oligonucleotide or backbone oligonucleotide.

[0055] However, in typical embodiments, the padlock, whether a gap-fill padlock or not, is provided as a single circularizable oligonucleotide.

[0056] In certain embodiments, a padlock probe does not have a secondary structure, and more specifically, does not contain an intramolecular double-stranded region or a stem-loop structure. However, a dumbbell probe, which does not have a secondary structure, is a specific subtype of a padlock probe. A dumbbell probe contains two stem-loop structures connected to each other, and one of the "loops" is neither closed nor open, but has free 5' and 3' ends that can be ligated. This "open loop" functions as the target-binding domain of the probe. The closed loop simply functions as a spacer connecting the ends of the duplex (stem). In other words, it can be seen as a padlock probe with a duplex region formed between the complementary sequences (regions) of the padlock. The duplex region functions as a signaling domain to which an intercalator can bind. Thus, the "open loop" of a dumbbell probe may contain a complementary target-binding region.

[0057] The padlock probe may contain one or more other regions or sequences that may be useful in the present methods. This may include, in particular, a detection sequence that allows the probe to be detected or identified. The detection sequence may, for example, be an identifying sequence, such as a barcode sequence, that can be detected to detect the padlock probe (more specifically, the complementary sequence of the detection sequence in the RCP can be detected). Alternatively, the detection sequence, or more specifically, its complementary sequence, may be detected using a detection oligonucleotide that hybridizes to the complementary sequence of the detection sequence in the RCP. Thus, the detection oligonucleotide is a detection probe, which is detected to detect the RCP, and the padlock probe used to generate it, thereby detecting the target nucleic acid molecule. The detection oligonucleotide may comprise a detection moiety, as described in more detail below. A complementary copy of the detection sequence in the padlock probe is generated in each repeating monomer of the RCP. This complementary copy in the RCP can hybridize to a detection oligonucleotide and, therefore, may itself be complementary to the detection oligonucleotide. Thus, it is understood that the detection sequence in the padlock probe can correspond to the sequence of the detection oligonucleotide used to hybridize to the RCP.

[0058] The target sequence detected in this method may contain one or more variant bases. Thus, it may be a single nucleotide variant, such as a single nucleotide polymorphism (SNP) or mutation, or it may contain two or more bases. Thus, the variant sequence may be composed of nucleotide moieties, two or more of which may be mutant bases. The mutant bases may be consecutive or non-consecutive.

[0059] The length of the target sequence is not critical and may vary depending on the context and the nature of the target molecule, the location or locus of the target sequence, or variant. Thus, by way of representative example only, the target sequence may be 1 to 10, e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 7, or 1 to 6 nucleotides in length. However, in certain embodiments, target sequences longer than one nucleotide may be beneficial to improve specificity; in such embodiments, the target sequence may be 2, 3, 4, 5, or 6, to 6, 7, 8, 9, 10, 12, 15, or 20 nucleotides in length. Thus, exemplary target sequences may be, for example, 4 to 10, 4 to 8, 4 to 7, 4 to 6, 5 to 10, 5 to 8, 5 to 7, or 6 to 8 nucleotides in length.

[0060] Silencing oligonucleotide is a linear oligonucleotide that comprises at least two, that is, at least first and second silencing regions.The only requirement is that the silencing region can hybridize with the target binding region of the padlock probe, which is located at the end of the padlock probe, for example, as shown in Figure 2, so that when hybridized with the silencing oligonucleotide, there is a gap of at least 5 nucleotides between the ends of the padlock probe.Therefore, the silencing region is separated in the silencing sequence by a gap sequence.

[0061] In particular, the gap sequence between the silencing regions may be at least 6, 7, 8, 9 or 10 nucleotides in length.

[0062] For example, the gap sequence between the silencing regions may be 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 5 to 40, 6 to 40, 7 to 40, 8 to 40, 9 to 40, 10 to 40, 5 to 30, 7 to 30, 8 to 30, 10 to 30, 5 to 25, 7 to 25, 8 to 25, 10 to 25, 5 to 20, 7 to 20, 8 to 20 or 10 to 20 nucleotides in length.

[0063] In certain embodiments, the silencing region may be located at the 5' and 3' ends of the silencing oligonucleotide, although this is not a requirement.

[0064] The silencing oligonucleotide may further comprise a silencing region corresponding to another sequence in the padlock probe, i.e., a sequence complementary to an internal sequence of the padlock probe, located between the two ends of the padlock probe. In particular, the silencing oligonucleotide may comprise a third silencing region. This is shown in Figures 2B and 2C and 8. The third silencing region is located between the first and second silencing regions in the silencing oligonucleotide. It is typically complementary to the silencing oligonucleotide binding site in the padlock probe, which may be located between the 5' and 3' ends of the padlock probe. Such a silencing oligonucleotide is referred to herein as a "triple binding" or "triple binder" and represents a particularly advantageous embodiment.

[0065] For example, when hybridized to a padlock probe, the presence of the third silencing region can prevent the hybridized 3' end of the padlock probe from being extended by a polymerase added for the RCA reaction.

[0066] Therefore, it will be understood that the third or further silencing region is located within the gap sequence. It may correspond to the entire gap sequence or a portion of the gap sequence. Thus, the gap sequence may be or include a sequence that is complementary to a sequence in the padlock probe that is not the target binding region of the probe. In such a configuration, the third silencing region may be complementary to any portion of the padlock probe that is not the target binding region. This silencing oligonucleotide binding site is usually located between the target binding regions at the 5' and 3' ends of the padlock probe or the backbone portion of a two-part padlock probe. The third silencing region may bind to the backpiece portion of the padlock probe or may be referred to as the backpiece silencing region.

[0067] In some embodiments, the third silencing region may hybridize to (e.g., be complementary to) all or part of the detection sequence of the padlock probe. In other words, the detection sequence may completely or partially overlap with the silencing oligonucleotide binding site that is complementary to the third silencing region of the silencing oligonucleotide.

[0068] Alternatively, if the silencing oligonucleotide does not contain any additional silencing regions in addition to the first and second silencing regions, the gap sequence is not complementary to any portion of the padlock probe. The gap sequence may be, for example, a random sequence.

[0069] As described above, the triple-binding configuration of the silencing oligonucleotide allows for a second round of RCA to be performed using the RCP generated in the first round (i.e., the "first RCP") as a ligation template for the padlock probe. This is demonstrated in Figure 7 and described in the Examples below. We have found that a triple-binding silencing oligonucleotide having first and second silencing regions (also referred to herein as the "arms" of the padlock probe) that bind to the first and second target-binding regions of the padlock probe and a third silencing region that binds to the backpiece of the padlock may disfavor the padlock from binding to the silencing oligonucleotide over the padlock binding to its target sequence. In other words, in such a configuration, the padlock probe may preferentially bind to its target sequence over the silencing oligonucleotide. In the absence of the target sequence, or when the padlock binding site in the target sequence is blocked or occluded, the silencing oligonucleotide binds to the padlock probe. However, in the presence of a target sequence (also referred to as a "template sequence"), the padlock probe binds to the target sequence. Without wishing to be bound by theory, it is hypothesized that this may be due to the padlock-target sequence binding structure being less rigid than the padlock-silencing oligonucleotide binding structure.

[0070] The target binding site (arm) of the padlock probe is complementary to the target sequence. The first RCP generated by RCA of the ligated padlock probe contains a monomer complementary to the padlock probe, and therefore, it can be seen that the first RCP contains repeated homologous copies of the target sequence within the repeat of the monomer. These repeat copies are therefore copies of the target sequence and provide binding sites for the padlock probe. In the presence of a ligase, the padlock probe preferentially binds to the target sequence, indicating that the padlock probe is ligated and serves as a template for a second round of RCA to generate a second-generation RCA product ("second RCP"). This results in signal amplification. The first RCP contains multiple repeated binding sites for the padlock probe, and therefore, multiple second RCPs can be generated. Such sRCA reactions are known in the art.

[0071] Those skilled in the art can readily design cognate silencing oligonucleotide / padlock probe pairs for use in RCA or sRCA reactions, if desired. Methods are known for designing oligonucleotides with different hybridization strengths, thereby achieving preferred hybridization for desired hybridization binding pairs.

[0072] The length of the silencing region is not critical as long as it is long enough to achieve specific hybridization with the complementary binding site in the padlock probe. It may also vary depending on the design and selected configuration of binding to the padlock probe. The first and second silencing regions bind to the target binding site of the padlock probe. They do not need to correspond exactly to the target binding site, but they must be long enough to specifically bind to those regions. The first and second silencing regions are usually at least 6 nucleotides long. The third silencing region may be shorter, for example, at least 2 nucleotides long. As described in the following examples, various lengths have been investigated and found to be effective.

[0073] In exemplary embodiments, the first and second silencing regions may be at least any one of 6, 7, 8, 9, and 10 nucleotides in length. The maximum length is not critical and depends on the length of the target binding portion of the padlock probe. For example, they may be up to any one of 7, 8, 9, 10, 11, 12, 15, 20, 25, or 30 or more nucleotides in length, as desired. Exemplary ranges include any range between any of the minimum or maximum integers listed above, such as 6 to 30, 6 to 20, or 6 to 18. Other ranges include 6 to 15, 6 to 12, 7 to 20, 8 to 20, 9 to 20, 10 to 20, etc. The third silencing region may be, for example, 2 to 50, 2 to 40, or 2 to 30 nucleotides in length.

[0074] Different combinations of the length of the first / second and third silencing regions can be used.For example, it has been shown that the first and second silencing regions of 18nt can be used in combination with the third silencing region of 20nt and function well, but this can also be changed to use with a shorter third silencing region.Similarly, the first and second silencing regions of 10nt also function well together with the third silencing region of 20nt.

[0075] As noted above, the first and second silencing regions need not correspond exactly in length to the target binding region of the probe, and thus the complementary binding site in the padlock probe for the silencing region may be a shorter or longer binding site for the target sequence of the padlock probe. A 10-12 nt long first or second silencing region may, for example, be shorter than the target binding site of the padlock probe, leaving a stretch of nucleotides free at the ends of the padlock probe (i.e., the 3'- and / or 5'-most nucleotides of the probe, i.e., those closest to the ligation site).

[0076] A silencing oligonucleotide is provided for each padlock probe used. In other words, each padlock probe has a cognate silencing oligonucleotide. Thus, for example, when different target-specific padlock probes, each containing a different target binding region, are used, each padlock probe has a different cognate silencing oligonucleotide. However, when a single or "common" padlock probe is used, only a single cognate silencing oligonucleotide is required.

[0077] To perform the method, a sample, or more specifically, a target nucleic acid, is contacted with a padlock probe. This contacting may involve other reagents. The sample, or a portion, fraction, or aliquot thereof, containing the target nucleic acid molecule may be contacted with the padlock probe. For example, in the case of in situ analysis or other actual analysis, such as a clinical sample, the sample may be directly contacted with the reagent. The sample may be pre-treated or processed prior to the contacting, for example, by fixation of a cell or tissue sample. In another embodiment, the target nucleic acid may first be separated or removed from the sample. Procedures for extracting or purifying nucleic acids, such as DNA, from various types of samples are well known in the art. For example, nucleic acids may be isolated from cells or from cell-free samples such as plasma. In some cases, it may also be desirable to fragment nucleic acid molecules. Techniques for this are known in the art and include, for example, specific digestion using nucleases, including restriction enzymes, or non-specific means such as shearing.

[0078] The contacting step with the padlock probe prepares the reaction mixture for the probe binding step. However, reagents for subsequent steps, e.g., an extension step if the padlock is a gap-fill padlock, may also be included. Furthermore, in alternative embodiments, or in addition, reagents for a ligation reaction may be included. Generally, the initial reaction mixture does not contain all of the reagents necessary to perform the RCA reaction, so that the RCA reaction can be controlled (i.e., initiated later). However, in some embodiments, it may be possible to include some RCA reagents (e.g., RCA primers or nucleotides) along with the padlock probe, or more generally, in the initial reagent mixture contacted with the sample.

[0079] To perform the probe binding step, the padlock probe is typically incubated with the target nucleic acid molecule. In the case of gap-fill padlocks, dNTPs and polymerase may also be included. Preferably, a ligase may also be included during the probe binding step. Reagents may be added to a single reaction mix or separately before or during the probe binding step. To allow probe binding, there may be an initial heating step, for example, to denature double-stranded nucleic acid molecules.

[0080] The reagents are typically provided in a buffer solution according to principles and techniques known in the art, for example, a buffer appropriate for the selected ligase enzyme may be selected.

[0081] The reaction mixture may be incubated under conditions suitable to promote or allow padlock probe binding (the so-called "annealing" step). If a preceding denaturation step is present, this may include a decrease in temperature. Conditions for these steps are known in the art and are within the ordinary skill of one of ordinary skill in the art to select or design. For example, room temperature or an annealing temperature in the range of 20-40°C may be used, e.g., 25-40°C or 25-37°C. In one embodiment, higher temperatures may be used, e.g., 50-65°C, e.g., 53-60°C or 55-60°C.

[0082] If a high annealing temperature is selected, the annealing temperature may be lowered for the extension step if a gap-fill padlock is used. Also, appropriate conditions may be selected according to those known in the art and the particular reagents, e.g., enzymes, used. For example, after the first annealing step, the temperature may be lowered to 28-40°C, e.g., 28-35, 30-35, 28-33, 30-33, 28-33, or 30-32°C.

[0083] The silencing oligonucleotide is contacted with the padlock probe before, during, or after the sample is contacted with the padlock probe, but before the RCA reaction occurs (ie, before the RCA reaction is initiated).

[0084] Preferably, the silencing oligonucleotide may be contacted with the sample simultaneously with the padlock probe. In this regard, the silencing oligonucleotide may be contacted separately or in a mixture. Thus, in some embodiments, a reagent mix may be prepared that includes the padlock probe and the silencing oligonucleotide. In other embodiments, the reagent mix may further include a ligase for the padlock probe ligation step. The mix may be contacted with the sample. Alternatively, the reagents may be added separately to the sample. The resulting reaction mixture is then incubated to allow binding and ligation of the padlock probe to occur.

[0085] The silencing oligonucleotides may be provided in a form that is pre-hybridized to a padlock probe, and such reagents are contacted with the sample or used to prepare the initial reagent mix.

[0086] In this regard, it should be understood that in formats in which the silencing oligonucleotide is pre-hybridized or added to the sample together with the padlock probe, the target nucleic acid will either remove the silencing oligonucleotide from the padlock or competitively inhibit the binding of the silencing oligonucleotide to the padlock probe. In other words, hybridization of the padlock probe to the target nucleic acid is prioritized over hybridization to the silencing oligonucleotide. However, any padlock probe that does not hybridize to the target molecule will remain hybridized to or become hybridized to the silencing oligonucleotide.

[0087] Alternatively, in another embodiment, a reagent mix containing a padlock probe and a ligase enzyme may be prepared, which may be contacted with the sample, and the padlock probe may be allowed to hybridize to the target nucleic acid. A silencing oligonucleotide may then be added. Any unbound padlock probe present in the reaction mixture may hybridize to the silencing oligonucleotide.

[0088] In another embodiment, a padlock probe may be contacted with a target nucleic acid and allowed to hybridize thereto. A ligase may then be added and any bound padlock probes may be ligated. A silencing oligonucleotide may then be added and hybridize to any unhybridized and unligated padlocks.

[0089] If the padlock is a gap-fill padlock, the initial reaction mix for padlock joining may also include the polymerase and nucleotides for the extension step. In such procedures, it may be desirable to include a heat inactivation step for the polymerase enzyme before RCA is initiated.

[0090] The padlock probe is ligated and circularized to generate a template for the first RCA reaction. Ligation is performed using the target nucleic acid molecule as a template. Any suitable ligase can be used, and representative ligases of note include, but are not limited to, temperature-sensitive ligases such as Splint® ligase (RBCV-1 DNA ligase or cholera virus DNA ligase), bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase, as well as thermostable ligases such as Taq ligase, Tth ligase, Ampligase®, Pfu ligase, and 9°N™ DNA ligase.

[0091] Suitable conditions for ligation are known in the art, and any necessary and / or preferred reagents may be combined into a reaction mixture and maintained under conditions sufficient for ligation. It will be apparent that ligation conditions may depend on the ligase enzyme used in the methods of the present invention. Thus, for example, Ampligase may be used and the temperature may be elevated for the ligation step. Alternatively, Splint® ligase may be used at room temperature.

[0092] If a temperature change or temperature control step is required, the method may be performed in a thermal cycling machine, which allows for easy control of temperature changes. However, an advantage of the method is that extreme temperature changes are not required; the method can be performed, for example, at room temperature, or, for example, at 20-37°C. The probe binding and ligation steps can be performed, for example, at room temperature.

[0093] Conditions for probe binding and ligation reactions can be optimized by routine experimentation according to principles known in the art, such that temperature, buffers, incubation times, gradient speeds, etc. can be adjusted to find optimal conditions.

[0094] Once the padlock probe is ligated, it is subjected to an RCA reaction. For example, if the method is performed in a heterogeneous or solid phase format, for example, if the sample is fixed or immobilized on a solid support, there may be an optional washing step before the RCA reaction. However, an advantage of the method is that a washing step is not required. Therefore, it is not preferred to include such a step. In some embodiments, the method proceeds to the RCA step without a washing step.

[0095] The RCA reaction is then initiated. This can be accomplished by adding a reagent to the reaction mixture to initiate the RCA reaction. This can be a complete RCA reaction mix containing all reagents necessary for the RCA reaction (e.g., polymerase, nucleotides, and optionally primers). Alternatively, one or more reagents can be added. For example, a single reagent can be added. Preferably, the RCA reaction is initiated by adding a polymerase. The polymerase is preferably added by adding an RCA reagent mix containing other reagents for the RCA reaction, such as nucleotides, in an appropriate buffer. However, in some embodiments, the other reagents for the RCA reaction can be added or pre-contained.

[0096] As mentioned above, the RCA reaction is well known in the art, and therefore the conditions for this step may be designed or selected according to known and literature protocols and principles. A strand-displacing polymerase enzyme, such as Phi29 or its derivatives, is used.

[0097] Primers for the RCA reaction may be added to the reaction mixture or may be pre-hybridized to the padlock probe. The binding site for the RCA primer may be provided in a region of the padlock probe (i.e., the padlock backbone region) that is distinct from the target binding region. In some cases, the target nucleic acid molecule may function as or provide the primer. If necessary, the 3' exonuclease action of a polymerase or a separate exonuclease may be used to digest the target nucleic acid to prepare a hybridized 3' end suitable for acting as an RCA primer for RCA of the circularized padlock probe.

[0098] The RCA reaction generates concatemeric RCA products (RCPs), which contain multiple repeat copies of the complementary sequence of the padlock probe, and the RCPs are detected to detect the target nucleic acid molecule.

[0099] The method may be carried out in a heterogeneous or homogeneous format, i.e. on a solid phase (or support), or in solution or suspension (i.e. without a solid phase and support), or indeed both, since a solid phase may be introduced at a later stage, e.g., in the step of detecting the RCP.

[0100] The format of the method may be selected based on the nature of the sample, or target nucleic acid molecule, or the desired reading or detection technique to be used. For example, a liquid-phase format may be employed for liquid, liquefied, or solubilized samples, or for isolated or purified nucleic acids, or for the detection of non-nuclear analytes in samples (e.g., detection of proteins in serum or plasma or other body fluids, etc.), or for the detection of any analyte in practical, convenient diagnostic or clinical testing.

[0101] On the other hand, to detect target sequences in solid samples, such as tissues or cells, a solid-phase format may be used, for example, in situ detection. For other reasons, such as a specific assay format or simply by choice, it may be preferable to immobilize the sample or target nucleic acid. Thus, an isolated cell sample may be immobilized on a support, or nucleic acid may be isolated or captured from the sample on a solid support, etc. In another embodiment, a primer for the second RCA reaction may be immobilized on a solid support.

[0102] In one embodiment, the method may be for localized detection of a target nucleic acid molecule, where "localized" detection means that the signal generated due to detection of the nucleic acid is localized to the nucleic acid, in this case, the RCP is localized to the target nucleic acid. Thus, the nucleic acid can be detected in the sample or at its location in the sample. In other words, the spatial location (or location) of the nucleic acid within the sample can be determined (or "detected"). This means that the nucleic acid may be localized to or within the cell in which it is located or expresses it, or to a location within a cell or tissue sample. Thus, "localized detection" can include determining, measuring, evaluating, or assaying the presence or amount and location or absence of the nucleic acid in any manner.

[0103] In certain embodiments, the method may be used for the localized, particularly in situ, detection of target nucleic acid sequences. More specifically, the method may be used for the localized, particularly in situ, detection of nucleic acids (particularly mRNA) in a sample of cells.

[0104] As used herein, the term "in situ" refers to the detection of a target nucleic acid sequence in its natural state, i.e., within a cell or tissue in which it normally occurs. Thus, it refers to the natural or natural location of a target nucleic acid sequence, e.g., RNA. Typically, the term refers to a nucleic acid being present within a cell or a sample of cells or tissue, e.g., its natural location within a cell or tissue and / or within its normal or natural cellular environment.

[0105] In other embodiments, as described above, detection is not localized or in situ. In still other embodiments, the method may be performed in solution. In particular, the nucleic acid may be in solution. Thus, for example, the method may be performed on a sample containing isolated nucleic acid.

[0106] The target nucleic acid molecule is present in a sample. The sample may be any sample containing any amount of nucleic acid from any source or origin, and it is preferred to detect the target nucleic acid molecule therein. The sample may be any clinical or non-clinical sample, and may be any biological, clinical, or environmental sample in which the target nucleic acid molecule may be present.

[0107] The sample may be any sample containing a target nucleic acid molecule, including both natural and synthetic samples, i.e., naturally occurring substances or manufactured preparations. Naturally occurring samples may be treated or processed before being subjected to the methods herein. All biological and clinical samples include, for example, any cell or tissue sample from an organism, or any body fluid or preparation 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. Samples may be freshly prepared, or they may be pre-treated in any suitable manner, for example, for storage.

[0108] Representative samples therefore include any material that may contain a target nucleic acid molecule, including, for example, food and industrial products, clinical and environmental samples. Samples may include any virus or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles. Thus, such biological material may include all types of mammalian and non-mammalian cells, plant cells, algae, including blue-green algae, fungi, bacteria, protozoa, etc., or viruses. Cells may be, for example, but are not limited to, human cells, avian cells, reptilian cells, etc.

[0109] Representative samples therefore include whole blood and plasma, serum and buffy coat, blood-derived products such as blood cells, urine, feces, cerebrospinal fluid or any other bodily fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, biopsy material, cell cultures, cell suspensions, conditioned medium or other samples of cell culture components, etc. Samples may be previously processed and prepared for use in the method in any convenient or desired manner, for example, by cell lysis or nucleic acid purification, isolation, etc.

[0110] In one embodiment, the sample comprises microbial cells or viruses isolated from a clinical sample or from a culture of a clinical sample, in such a sample, the target nucleic acid molecule may be a nucleotide sequence present in the microbial cells or viruses, for example, a nucleotide sequence that is characteristic or discriminative or identifying for the microbial cells or viruses at any level, for example, type, group, class, genus, species or strain level.

[0111] In another embodiment, the sample may contain cell-free DNA. The sample may be a sample such as plasma or serum that directly contains cell-free DNA, or the cell-free DNA may be isolated. The cell-free DNA may include circulating tumor DNA.

[0112] In the case of localized in situ detection, the sample may be any sample of cells that can produce nucleic acid molecules, to the extent that such a sample can be examined by localized in situ detection. It may also be a sample that is fixed and present in a detectable or visualized location in the sample. Thus, the sample is any sample that reflects the normal or natural ("in situ") location of a nucleic acid, e.g., RNA, i.e., any sample in which it normally or naturally occurs. Such a sample preferably includes or comprises a group of cells, such as a cell or tissue. Examples include samples such as cultured, harvested, or biopsied cell or tissue samples in which nucleic acids can be detected to reveal the qualitative nature of the nucleic acid, i.e., its presence or nucleotide sequence, or the presence and / or identity of one or more nucleotides in the nucleic acid, as well as its location relative to other cellular characteristics. Cell samples may be freshly prepared or may be pre-processed in any suitable manner, such as by fixation or freezing. Thus, fresh, frozen or fixed cells or tissues, such as FFPE tissue (Formalin Fixed Paraffin Embedded), may be used. The sample may contain any cell type that contains nucleic acid, including all types of cells, as described above.

[0113] Samples may also be treated to immobilize nucleic acids, such as RNA, contained in cells, to the sample, e.g., to immobilize it to a cellular matrix. Such techniques are known and described in the art. For example, in the field of in situ hybridization, reagents for immobilizing mRNA to cells are known. In particular, the 5' phosphate group in RNA can be linked to amines present on proteins in the cellular matrix by EDC-mediated conjugation (EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), thereby contributing to maintaining the localization of the RNA relative to other cellular components. Such techniques have previously been described in connection with microRNAs and their detection by in situ hybridization. Alternatively, in procedures involving a reverse transcription step to generate cDNA from an RNA target, the 5' ends of the cDNA primers and / or cDNA molecules may be subjected to cross-linking activity. This may be achieved, for example, using DSP chemicals or acrylic acid NHS ester.

[0114] The sample need not be a sample naturally containing nucleic acids or a source of nucleic acids (e.g., cells, viruses, biological or clinical material, etc.), since the target nucleic acid molecule need not itself be the target analyte of the assay, but can be, for example, a reporter molecule used or generated during an assay for any desired analyte. As noted above, the sample may be a synthetic or artificial sample. Thus, it may be a sample that has been subjected to a detection assay for an analyte from which the target nucleic acid was generated or to which the target nucleic acid molecule was added. It may also be a reaction mixture or a reaction product, e.g., a product resulting from an immunoassay such as immuno-PCR, immuno-RCA, or a proximity assay (e.g., proximity ligation assay (PLA) or proximity extension assay (PEA)) for detecting a target analyte, as described above.

[0115] A target analyte may be any analyte for which detection is desired. As noted above, in embodiments, the target nucleic acid molecule of the methods herein is the target analyte. In other embodiments, when the target nucleic acid molecule is a reporter, the target analyte may be any analyte for which detection is desired. An analyte may be a nucleic acid, a protein (which term includes peptides and polypeptides), or any other chemical or biological molecule or moiety, including, for example, carbohydrates (e.g., those that can cause glycosyl groups on proteins). Thus, a target analyte may be, for example, a modified protein with a post-translational modification that is detected in an assay for the analyte.

[0116] In some embodiments, the target analyte may be a protein or proteinaceous molecule component detected on the surface of a cell, or a vesicle, or other cellular or intracellular compartment. For example, extracellular vesicles or exosomes may be detected and distinguished by the different proteins present on their surface.

[0117] As mentioned above, padlock probes may contain one or more additional sequences that act to introduce a sequence into the ligated product and thereby into the RCP (as a complementary copy). This may be, for example, a detection sequence, such as a barcode or identification motif, or a binding site for a detection probe or primer. Such additional sequences may be found, for example, in the backbone region of the padlock probe, i.e., part of the region between the target binding regions. In dumbbell probes, they may be in the duplex region of the probe. Tags such as barcodes or probe / primer binding sites may be designed with different needs / purposes, for example, to introduce universal or common sequences, such as binding sites for universal or common amplification primers, so that different ligated probes can be processed together, for example, in a multiplex setting. This allows different ligated probes to be amplified together, for example, in library amplification by PCR or RCA.

[0118] In particular, a padlock probe may contain a detection sequence that can be detected by it. The complementary sequence of the detection sequence is incorporated into the RCP and can be detected, for example, by the detection probe binding thereto or by sequencing. The detection sequence may be specific to the padlock probe and therefore to the target sequence or sequence variant desired to be detected. Thus, each padlock probe may have a different detection sequence. The detection sequence may be detected to detect or identify which padlock probe was amplified in RCA and therefore which target sequence was present. Such a protocol may be applied, for example, in a method for detecting target sequence variants, where each padlock is provided with a detection sequence specific to a particular variant. In this way, the detection sequence may be understood as a marker or identification sequence. As used herein, the term "detection sequence" includes both the detection sequence present in the padlock probe and its complementary copy as it appears in the RCP.

[0119] Thus, detection sequences may be used to "tag" or mark different padlock probes so that they, or their ligation or amplification products, can be easily distinguished from one another. Additionally, such sequences may be used to tag different samples, etc., so that they may be pooled (i.e., "sample" tags or markers). Thus, in a multiplex setting, different probes (i.e., probes for different target nucleic acid sequences or different variants) may be tagged with different tag sequences (e.g., different marker or detection sequences) and / or may be tagged with the same tag sequence(s), e.g., for the introduction of a common or universal sequence. Such methods may be used, for example, in multiplex detection of multiple target nucleic acids in a sample, in conjunction with specific detection methods, including the use of detection probes or sequencing methods, e.g., sequencing by hybridization, sequencing by ligation, or other next-generation sequencing chemistries.

[0120] As used herein, the term "hybridization" or "hybridizing" refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex through Watson-Crick base pairing or any similar base pairing interaction. Two nucleotide sequences are "complementary" to one another if the molecules share base pairing homology. Thus, a complementary region in a molecule, probe, or sequence refers to the portion of the molecule, probe, or sequence that can form a duplex. Hybridization does not require 100% complementarity between sequences; therefore, regions that are complementary to one another do not require, but do not exclude, perfect sequence complementarity. Thus, complementary regions may contain one or more mismatches. Therefore, "complementary," as used herein, refers to "functionally complementary," i.e., a level of complementarity sufficient to achieve effective hybridization, which includes degrees of complementarity less than 100%. The degree of mismatch allowed can be controlled by appropriate adjustment of hybridization conditions. Those skilled in the art of nucleic acid technology can empirically determine duplex stability, taking into account several variables, including, for example, the length of each molecule or probe oligonucleotide, the base pair composition, the ionic strength, and the incidence of mismatched base pairs, and following guidance provided in the art. Thus, the design of appropriate probes, their binding regions, and the conditions under which they hybridize to their respective targets is well within the ordinary skill of one of ordinary skill in the art.

[0121] For example, the region of complementarity to the target sequence in the binding region of a padlock probe, or between the detection sequence and detection oligonucleotide, or between an RCA primer and a circularized padlock probe, may be at least 6 nucleotides long, or more specifically, at least 7, 8, 9, or 10 nucleotides long, to ensure specificity of binding. The upper length limit of the region is not critical, but may be, for example, 50, 40, 35, 30, 25, 20, or 15 nucleotides. Thus, the complementary region may have a length ranging between any one of the lower length limits and the upper length limit. In the case of padlock probes, the length of each target binding region may be shorter, so that the combined length of the two binding regions when hybridized to their targets is longer. For example, each target binding region may be 8 to 15, e.g., 10 to 12, nucleotides long, resulting in a combined hybridized length of 16 to 30, e.g., 20 to 24, nucleotides long. Within the constraints of probe conformation and domain spacing and desired or preferred hybridization, it may be preferable to minimize the total length of the padlock probe to minimize the size of the circle subjected to RCA, and therefore minimize the length of the complementary region, where possible.

[0122] RCPs can be detected using any suitable protocol. Depending on the target molecule to be detected, the purpose of the method, and / or the specific details of the procedures used in the method, the detection protocol used may non-specifically or specifically detect RCPs.

[0123] For example, RCPs can be directly detected, for example, by cleaving the concatemers to generate monomers that can be detected using gel electrophoresis, or more commonly, by hybridizing a labeled detection oligonucleotide that hybridizes to the detection sequence in the RCP, as described above. However, the detection oligonucleotide does not need to be directly labeled. For example, the detection oligonucleotide can be an unlabeled probe that functions as a sandwich probe. The concept of sandwich probes is well known in the art and can be applied according to any convenient protocol. Sandwich probes can bind to RCPs, but they themselves are not directly labeled. Instead, they contain a sequence to which a labeled secondary oligonucleotide can bind, thus forming a "sandwich" between the RCP and the labeled secondary oligonucleotide.

[0124] RCPs can also be detected using sequence-nonspecific nucleic acid labeling methods, such as DNA-binding stains or dyes, which are widely known in the literature, or by using labeled nucleotides for incorporation into RCPs. Alternatively, RCPs can be detected indirectly, for example, by amplifying the product by PCR and detecting the amplified product.

[0125] RCPs may be detected using any of the well-established methods for the analysis of nucleic acid molecules known in the literature, including liquid chromatography, electrophoresis, mass spectrometry including CyTOF, electron microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric assays such as ELISA, flow cytometry, mass spectrometry, or turbidimetric, magnetic, particle counting, electrical, surface sensing, and gravimetric detection techniques. Generally speaking, such techniques are associated with solution-based assays.

[0126] However, advantageously, RCPs can generally be detected using labeled detection oligonucleotides, i.e., detection oligonucleotides prepared with a detection moiety. A detection moiety is any moiety that can be detected, i.e., can directly or indirectly generate a detectable signal. Thus, a detection moiety can be considered as any detectable label that can directly or indirectly generate a signal. For example, a detection moiety can be spectroscopically or microscopically detectable, for example, it can be a fluorescent or colorimetric label, a particle, e.g., a bead, or an enzymatic label. Any label used in immunohistochemistry techniques can be used.

[0127] In multiplex procedures for detecting different and / or variant target sequences, different RCP products can be detected and distinguished by, for example, in situ sequencing, including sequencing-by-synthesis, sequencing-by-hybridization, sequencing-by-ligation, next-generation sequencing, and / or sequential barcode decoding techniques, and / or by using detection probe oligonucleotides. Depending on the level of multiplexing, multiple labeling methods may be used, for example, via differential labeling with fluorescently or spectrophotometrically detectable oligonucleotides, according to techniques well known in the art. Such differentially labeled detection probes can be used during flow cytometry or microscopic detection techniques, such as imaging, to detect a large number of sequences; for example, combining at least two fluorescent dyes at different ratios can lead to the generation of multiple populations of fluorescent labels.

[0128] In methods involving the use of detection oligonucleotides, the detection oligonucleotide or any secondary labeled probe may be directly or indirectly labeled with a detectable label. A directly detectable label is one that can be detected directly without the use of additional reagents, whereas an indirectly detectable label is one that becomes detectable by using one or more additional reagents, e.g., the label is a component of a signal producing system composed of two or more components. In many embodiments, the label is a directly detectable label, including, but not limited to, fluorescent labels, colored labels, radioisotope labels, chemiluminescent labels, and the like. In many embodiments, the label is a fluorescent label, and the labeling reagent used in such embodiments is a fluorescently tagged nucleotide, such as fluorescently tagged CTP (e.g., Cy3-CTP, Cy5-CTP). Fluorescent moieties that can be used to tag nucleotides to produce labeled probe nucleic acids (i.e., detection probes) include, but are not limited to, fluorescein, cyanine dyes such as Cy3, Cy5, Alexa555, Bodipy630 / 650, etc. Other labels, such as those listed above, can also be used as known in the art.

[0129] For liquid-phase detection, the detection moiety may be a colored bead, which can be easily visualized. Such beads, for example, colored polystyrene beads, are widely available.

[0130] Although various detection methods can be used, preferably, RCP can be detected by visualization, including microscopy or flow cytometry.In either case, directly or indirectly labeled detection oligonucleotides can be used, for example, using fluorescent or colored labels that can be easily detected.In this regard, the label can include beads or other detectable particles.In microscopy-based methods, RCP can be detected by imaging.

[0131] As described above, kits for carrying out the methods are also provided herein. The kits may include the padlock probes and silencing oligonucleotides described above, optionally along with one or more reagents and / or instructions for use of the kit. Such reagents include dNTPs and polymerase and ligase enzymes, as well as RCA primers for the RCA reaction. Further components may include buffers or other reaction components for one or more of the various reactions. Still further optional components may include means or reagents for detecting the RCP. This may include, for example, detection oligonucleotides and any necessary secondary labeling reagents, including, for example, those described above. Further optional components may include a solid support and / or means for capturing and / or immobilizing target nucleic acid molecules or reaction components. The instructions may be, for example, in printed form, on a computer-readable medium, or as a website address.

[0132] As mentioned above, the method may be carried out using a solid phase, for example, where the RCA product is immobilized on a solid phase, which may result from the immobilization of a target molecule, for example, in an in situ detection technique.

[0133] Immobilization of the first RCA product and / or target molecule on a solid phase can be achieved in various ways, according to principles well known in the art. Accordingly, several embodiments of solid-phase assays are envisioned. In one such embodiment, a sample may be provided on a solid support, for example, in situ. Alternatively, the target nucleic acid molecule may be captured by an immobilized (or immobilizable) capture probe, and the RCA product may be generated such that it is bound to the analyte, for example, by a primer for RCA that is the target molecule or that binds to the target molecule. Alternatively, the RCA product may simply be immobilized on a solid support. For example, the primer for the first RCA may be provided with an immobilizable group or moiety or immobilization means, or may be immobilized prior to the first RCA.

[0134] As noted above, the target nucleic acid may itself be immobilized (or immobilizable) on a solid phase, for example, by non-specific absorption. In certain such embodiments, the molecule may be present within a cell, optionally fixed and / or permeabilized, which is bound (or may be bound) to a solid support, for example, a tissue sample containing the target molecule may be immobilized on a microscope slide.

[0135] The advantages of the method herein are as described above. Such advantages are particularly beneficial in that they facilitate the performance of in-solution assays. By avoiding the need for washing steps or exonuclease removal of unhybridized probes, a simplified assay protocol can be provided. In particular, the method can be easily performed in a single reaction vessel, e.g., a single tube or reaction well. Thus, the method can be performed quickly, in less than a few minutes, by adding reagents to the reaction vessel. Furthermore, RCPs can be detected in a single vessel using, for example, detection oligonucleotides provided with easily visualized detection moieties, such as colored beads. In this manner, a direct visual readout can be obtained, for example, to detect the presence or absence of a target nucleic acid.

[0136] Such methods lend themselves to clinical diagnostic assays, including the advantageous use of diagnostic assays for home testing or field use, without the need for a clinical diagnostic laboratory.

[0137] By way of example, the methods can be employed to test clinical samples for the presence of pathogens, e.g., by detecting the presence or absence of nucleic acid of the pathogen. This may include, inter alia, testing samples such as saliva or nasal or oral swabs for the presence of viruses, including SARS-CoV2.

[0138] Furthermore, the technique is rapid, has minimal equipment requirements, and allows for multiplexed analysis of sequence variants with enhanced sensitivity. [Example]

[0139] The method will now be described in more detail in the following non-limiting examples. [Example]

[0140] Models

[0141] method 1) Synthetic templates representing the target SARS-CoV-2 genome containing nine different target sequences were prepared. The templates were added to tubes at a concentration of 10 pM in a volume of 20 μl. Control tubes were prepared without template but instead containing the same amount of "incorrect template" (i.e., synthetic non-target template molecules, representing "negative sample tubes").

[0142] 2) A ligation mix was prepared by mixing 5 nM of each of nine different padlock probes targeting different sequences in the target genome, 1–5 nM of each silencing oligonucleotide (one silencing oligonucleotide per padlock), and ligase. Five microliters of the ligation mix was added to the template. The tube was incubated at room temperature for 5 minutes. 1. Alternative 1. Mix padlock and ligase and add to template. Add silencing oligonucleotide and incubate for 5 minutes at room temperature. 2. Alternative 2. Add the padlock to the template, followed by the ligase, followed by the silencing oligonucleotide. Incubate for 5 minutes at room temperature.

[0143] 3) The RCA reaction was performed. The components for RCA were prepared by adding detection beads and Phi29 polymerase to an RCA mix containing BSA, Phi29 buffer, and dNTPs. 15 μl of the RCA mix was added to the ligation reaction, and the tube was incubated for 25 minutes at 37°C. The detection beads were functionalized colored beads carrying detection oligonucleotides.

[0144] 4) The results were read by visual inspection of the tubes.

[0145] result As shown in Figure 3, the sample tube containing the synthetic target template reported a positive result (top panel). A positive result could be confirmed by visualization of the clearly colored RCA reaction product at the bottom of the tube (resulting from the aggregation of colored detection beads in the RCP). Tubes that did not contain the synthetic target template (i.e., contained an incorrect template) reported a negative result; no aggregation of colored beads by the RCA reaction product was observed, and the colored detection beads could be seen evenly distributed throughout the reaction solution in the tube.

[0146] To demonstrate the reproducibility of this method, we repeated the experiment comparing synthetic templates (four replicates) representing the correct target nucleic acid molecule with control templates (four replicates) lacking the target sequence for the padlock probe representing the incorrect target molecule. The results are shown in Figure 4A. Positive results can be confirmed for the correct target molecule, and negative results can be confirmed for the incorrect target molecule. This demonstrates that the specificity of the detection assay can be controlled using silencing oligonucleotides.

[0147] The results of titrating different amounts of silencing oligonucleotide are shown in Figure 4B. This shows that clearly distinct positive reactions can be visualized at silencing oligonucleotide concentrations of 5, 4.5, 4, and 3.5 nM. RCA reaction products are still visible at 3 nM, but are less clear at 2.5 nM. Clear RCA reaction products are not visible at 2 and 1.5 nM silencing oligonucleotides. This demonstrates that the presence of a sufficient amount of silencing oligonucleotide allows visualization of the RCA reaction products. This indicates that those skilled in the art will be able to easily determine the appropriate ratio between padlock:silencing oligonucleotide and template. [Example]

[0148] Testing of clinical samples

[0149] method 1) Clinical samples confirmed positive or negative for COVID-19 by PCR were obtained. Samples were diluted appropriately in the carrier buffer in which they were provided and prepared. 20 μl aliquots of sample were added to tubes. Tubes 1-20 contained samples that were PCR positive for COVID-19, and tubes 21-40 contained samples that were PCR negative for COVID-19.

[0150] 2) A ligation mix was prepared by mixing 5 nM of each of nine different padlock probes targeting different sequences in the target genome, 1-5 nM of each silencing oligonucleotide (one silencing oligonucleotide per padlock), and ligase. Five microliters of the ligation mix was added to the template. The tube was incubated at room temperature for 5 minutes. For comparison, the silencing oligonucleotide was omitted from the ligation mix. 1. Alternative 1. Mix padlock and ligase and add to template. Add silencing oligonucleotide and incubate for 5 minutes at room temperature. 2. Alternative 2. Add the padlock to the template, followed by the ligase, followed by the silencing oligonucleotide. Incubate for 5 minutes at room temperature.

[0151] 3) The RCA reaction was performed. The components for RCA were prepared by adding detection beads and Phi29 polymerase to an RCA mix containing BSA, Phi29 buffer, and dNTPs. 15 μl of the RCA mix was added to the ligation reaction, and the tube was incubated for 25 minutes at 37°C. The detection beads were functionalized colored beads carrying detection oligonucleotides.

[0152] 4) The results were read by visual inspection of the tubes.

[0153] result Figure 5 shows the results of experiments without (A) and with (B) silencing oligonucleotides in the ligation mix. In the absence of silencing oligonucleotides, no difference can be seen between the COVID-19-positive tubes (tubes 1–20) and the COVID-19-negative tubes (tubes 21–40). In contrast, in the presence of silencing oligonucleotides, a positive result (presence of RCA reaction products) can be detected in the COVID-positive tubes. For tubes 17–20 in (B), the results were not as clear as those in the other tubes, but aggregation of colored beads was visible, indicating the presence of RCA reaction products, unlike tubes 21–32. [Example]

[0154] Demonstration of improved sensitivity using model systems

[0155] Experiments were performed using the model system of Example 1 with and without silencing oligonucleotides.

[0156] The results are shown in Figure 6, which demonstrates that the use of triple-linked silencing oligonucleotides results in improved sensitivity. (A) Titration of template oligonucleotides in the model system without silencing oligonucleotides. (A) Template oligonucleotides at concentrations of 5 nM, 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, and a negative control (Neg C) demonstrate binding of different detection oligonucleotides to either rolling circle products (RCPs) or free padlocks; in this experiment, interaction with RCPs is not inhibited by the silencing oligonucleotides. The detection limit here is between 5 pM and 50 pM. (B) Titration of template oligonucleotides in the model system with triple-linked silencing oligonucleotides. (B) shows the template oligonucleotide and negative control (Neg C) at concentrations of 5 nM, 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, and 5 fM, together with the silencing oligonucleotide at a 1:1 ratio relative to the padlock. This demonstrates that the RCP can grow without interference from a free padlock that is bound by the silencing oligonucleotide at one arm of the growing padlock. If the padlock is able to bind at one of its arms, the RCP becomes double-stranded. Only complete binding of the padlock to the growing RCP is possible (because the padlock prefers binding to the complete target (18 + 18 nt (36 nt)) over the triple-binding silencing oligo. However, if the free padlock binds to the RCP at one arm, the silencing oligo then binds to the other arm, and Phi29 cannot make the product double-stranded). This then allows for second-generation RCP. The silencing oligonucleotide allows complete binding of the padlock to the template, which improves the sensitivity of the assay to a detection limit between 5 fM and 50 fM. [Example]

[0157] Demonstration of second-round RCA using triple-binding silencing oligonucleotides

[0158] Using the model system of Example 1, two different silencing oligonucleotide designs were compared: a silencing oligonucleotide containing only the first and second silencing regions (targeting the padlock probe target binding site / arm), separated by a 20 nt spacer sequence, and a triple-binding silencing oligonucleotide that also contained a third silencing region.

[0159] Silencing oligonucleotides were tested with template molecules at different concentrations ranging from 5 nM to 5 fM.

[0160] The results are shown in Figure 7, parts (A) and (B), respectively. As can be seen in (B), in the presence of triple-binding oligonucleotides, a larger amount of RCA product was observed, including that of the template molecule at a lower concentration, compared to the double-binding silencing oligonucleotides in (A).

[0161] The results show that triple-linked silencing oligonucleotides with three silencing regions enable a second round of RCA (after the first round). This can only occur if the silencing oligonucleotide binds to the padlock at three positions due to the more rigid conformation of the padlock probe / silencing oligo cognate, so that if the correct template sequence is present, the padlock prefers to bind to the template over the silencing oligo. Silencing oligonucleotides (A) that do not bind to the padlock backpiece do not form this more rigid conformation when binding to the padlock probe and therefore have no preference for the template sequence. Therefore, (A) allows for lower sensitivity and does not enable a second round of RCA. Triple-linked silencing oligonucleotides allow complete binding of the padlock to the template—first, the template target molecule (e.g., a viral genome), and second, the growing RCP containing the target sequence from the first target molecule—which improves the sensitivity of the assay. [Example]

[0162] Examination of differently designed triple-binding silencing oligonucleotides

[0163] Two designs of silencing oligonucleotides, designated Danish conformation (BD) and omega conformation (BO), respectively, were tested in the model system of Example 1, as depicted in FIG.

[0164] Different lengths of the first, second, and third silencing regions were tested, ranging from 6 to 18 for the first and second silencing regions ("arms"), and from 2 to 30 for the third silencing region ("backpiece," Bp).

[0165] The results are shown in Table 1 below.

[0166] [Table 1]

[0167] The results can be summarized as ΔG(pad - temp) < ΔG(pad - block) < GΔ(pad) - 56 < ΔG(pad - block) < -17.

[0168] Silencing oligonucleotides having 18 - 20 - 18 or 10 - 20 - 10 first - second - third silencing regions (where the 10 nt first and second silencing regions leave the target binding site of the padlock closest to the ligation site free) are very efficient.

[0169] The efficiency of other variants is lower, but nevertheless they function efficiently enough.

Claims

1. 1. A method for detecting a target nucleic acid molecule in a sample, the method comprising: (i) contacting the sample with a padlock probe that includes target binding regions at its 5' and 3' ends that are complementary to probe binding sites in the target nucleic acid molecule; (ii) contacting the padlock probe with a silencing oligonucleotide comprising first and second silencing regions complementary to the target binding regions at the ends of the padlock probe, wherein the two silencing regions are separated by at least 5 nucleotides, said contacting occurring before, during or after contacting the sample with the padlock probe; (iii) juxtaposing and hybridizing the target-specific binding regions of the padlock probe to the target nucleic acid molecule for direct or indirect ligation to each other; (iv) directly or indirectly ligating the 5' and 3' ends of the padlock probe to circularize the padlock probe; (v) after the silencing oligonucleotide has contacted the padlock probe, contacting the sample containing the circularized padlock probe with a polymerase and performing an RCA reaction using the circularized padlock probe as a template for RCA to generate an RCA product (RCP); (vi) detecting the RCP to detect the target nucleic acid sequence.

2. The method of claim 1 , wherein the method is carried out in solution.

3. 3. The method of claim 1 or claim 2, wherein the target nucleic acid molecule is a target analyte, or is generated from a target nucleic acid analyte, or is a reporter for a target analyte.

4. 4. The method of claim 1, wherein the padlock probe is pre-hybridized to the silencing oligonucleotide, and the target nucleic acid molecule removes the silencing oligonucleotide from the padlock probe hybridized to the target nucleic acid molecule.

5. 4. The method of claim 1, wherein the padlock probe and the silencing oligonucleotide are contacted simultaneously or in a mixture with the sample containing the target nucleic acid molecule.

6. 4. The method of claim 1, wherein after hybridizing the padlock probe to the target nucleic acid molecule or after the ligation step (iv), the silencing oligonucleotide is contacted with the sample so that it hybridizes to any unbound or unligated padlock probe in the sample.

7. 7. The method of claim 1, wherein the silencing region is located at the 5' and 3' ends of the silencing oligonucleotide.

8. 8. The method of claim 1, wherein the silencing oligonucleotide comprises a third silencing region between the first and second silencing regions that is complementary to a silencing oligonucleotide binding site in the padlock probe, and the silencing oligonucleotide binding site is located inside the padlock probe, between the 5' and 3' ends of the padlock probe.

9. 9. The method of any one of claims 1 to 8, wherein the padlock probe comprises a detection sequence, which allows for detection of the padlock probe, or an amplicon or reverse complement copy thereof.

10. 10. The method of claim 9, wherein the detection sequence is a binding site for a detection oligonucleotide or comprises a barcode sequence.

11. The method of claim 10 , wherein the detection oligonucleotide is linked to a detection moiety.

12. 12. The method of claim 11, wherein the detection moiety is a bead, a fluorescent or colorimetric label, a dye, or an enzyme substrate.

13. 13. The method of claim 9, wherein the detection sequence partially or completely overlaps with the silencing oligonucleotide binding site that is complementary to the third silencing region of the silencing oligonucleotide.

14. 14. The method of any one of claims 1 to 13, wherein the method is performed in multiplex to detect two or more target nucleic acid molecules in the sample, and step (i) comprises contacting the sample with two or more padlock probes, each specific for a different target nucleic acid, and wherein a different silencing oligonucleotide is provided for each different padlock probe.

15. 15. The method of any one of claims 1 to 14, wherein the method is carried out in a single reaction vessel.

16. 1. A kit for detecting a target nucleic acid molecule in a sample, said kit comprising: (i) a padlock probe comprising target binding regions at its 5' and 3' ends that are complementary to probe binding sites in the target nucleic acid molecule; (ii) a silencing oligonucleotide comprising first and second silencing regions complementary to the target binding regions at the ends of the padlock probe, wherein the two silencing regions are separated from each other by at least 5 nucleotides.

17. 17. The kit of claim 16, wherein the padlock probe and / or the silencing oligonucleotide is as defined in any one of claims 7 to 13.

18. 18. The kit of claim 16 or claim 17, wherein the kit comprises two or more different padlock probes, each specific for a different target molecule, and two or more silencing oligonucleotides, each specific for a different padlock probe.

19. A silencing oligonucleotide for blocking the target binding region of a padlock probe, wherein the silencing oligonucleotide comprises first and second silencing regions complementary to the target binding regions at the ends of the padlock probe, the two silencing regions being at least 5 nucleotides apart from each other, and the silencing oligonucleotide further comprises a third silencing region located between the first and second silencing regions and complementary to a silencing oligonucleotide binding site in the padlock probe, the third silencing region being located between the target binding regions of the padlock probe.

Images