Method for detecting target nucleic acid sequence

Abstract

The invention provides a method for detecting a target nucleic acid molecule in a sample by using a lock-on probe and rolling circle amplification. The methods herein provide a novel method of removing free padlock probes that fail to hybridize or link to a target and involve the use of "silent" oligonucleotides to "block" such unbound or unlinked padlock probes.

Description

Technical Field

[0001] The present disclosure and invention relate to the field of nucleic acid detection. Specifically, the present disclosure and invention relate to a method for detecting a nucleic acid target molecule using a padlock probe and rolling circle amplification (RCA). The method described herein provides a novel method for removing free padlock probes that fail to hybridize or connect to their targets, and involves using an oligonucleotide called a silencing oligonucleotide to "block" such unbound or unconnected padlock probes. Silencing oligonucleotides and kits for the method are also provided. Background Art

[0002] The detection of target nucleic acid molecules has applications in many different fields, including, in particular, in the diagnosis, prognosis and / or treatment of diseases in the clinic, as well as in the fields of research and biosafety. In addition, nucleic acids are often used as labels or markers in reporter systems for detecting other analytes, wherein nucleic acids are detected as surrogates or indicators of related analytes.

[0003] Target nucleic acid sequences can be easily detected using labeled hybridization probes, but simple hybridization probes have relatively high detection limits and are not easily used to distinguish similar nucleic acid sequences. To increase sensitivity, target nucleic acid molecules containing the target sequence are often amplified to increase the amount of target sequence available for detection. RCA is commonly used for such amplification and is often used in conjunction with padlock probes.

[0004] RCA utilizes a strand-displacing polymerase and requires a circular amplification template, which can be provided by a circularized padlock probe. Amplification of the circular template provides a concatemer of RCA products containing multiple copies of a sequence complementary to the amplification template sequence. Such concatemers typically form a globular or "spot" structure that can be easily visualized and detected, and thus RCA-based assays have been widely used for the detection of nucleic acids and, indeed, more generally as reporter systems for the detection of arbitrary target analytes.

[0005] Padlock probes are typically linear oligonucleotides with two separate target-complementary binding regions connected by a central "backbone" region. Once the probe binds (hybridizes) to its target nucleic acid sequence, the ends of the probe can be ligated together, thereby circularizing 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 many detection methods used today. 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 because only probes that base pair correctly at the ligation site will ligate, thereby generating a template for the molecule to be detected. The ends of the padlock probes can be directly ligated to each other when the target binding regions of the padlock probes adjacent to each other hybridize directly to the target nucleic acid sequence. Alternatively, the target binding regions of the padlock probes may have a gap between them and the target nucleic acid upon hybridization, which can be filled by hybridizing one or more gap oligonucleotides in the gap region, or by polymerase-catalyzed extension of the hybridized 3' end of the probe. In this way, the hybridized ends of the padlock probes can be indirectly ligated to each other, i.e. they each hybridize to the central "gap sequence". This "gap-filling" padlock probe is also called a molecular inversion probe.

[0006] In general, in RCA-based methods using padlock probes, it is often desirable to remove unbound padlock probes, i.e. padlock probes that are not hybridized to their target nucleic acid (i.e. free padlock probes) or probes that are not connected before the RCA reaction is performed. In this way, mis-priming of free padlock probes in the reaction mixture can be avoided, for example by hybridization with the product of the RCA reaction. More specifically, free padlock probes that subsequently hybridize with the RCA product (RCP) generated in the RCA reaction can trigger an extension reaction with the RCP as a template, resulting in the production of a double-stranded product, to which the detection oligonucleotide used to detect the RCP cannot bind. By removing or "neutralizing" such free padlock probes so that they cannot participate in subsequent hybridization or extension reactions, the specificity and / or sensitivity of the method can be improved.

[0007] Current measures to remove free padlock probes include washing. However, this approach requires a solid phase and is therefore not suitable for homogeneous or so-called "in-solution" methods, which do not use a solid phase. Free padlock probes can also be removed by enzymatic digestion, most commonly using exonucleases, which are able to digest nucleic acids with free or unligated ends, but will not digest circularized padlock probes without free ends. However, the disadvantage of this approach is not only the cost of the additional enzyme, but also the need to inactivate the exonuclease to prevent it from digesting other detection / reaction components (including, for example, the RCP). This inactivation is typically achieved by heating to 95°C. In order to be able to run the assay in a single tube in solution without the need for additional enzyme catalysis steps, heating steps or washing steps, an alternative solution is needed. Summary of the invention

[0008] The present inventors have proposed an alternative solution to solve the problem of removing unwanted unhybridized padlock probes in detection methods based on padlock probes and rolling circle amplification (RCA).

[0009] This involves the use of an oligonucleotide, called a "silencing oligonucleotide," which is able to hybridize to the ends of the padlock probe, thereby preventing it from participating in further reactions. In effect, the padlock probe becomes "closed" and functionally inactive unless the correct template is present. In other words, the padlock probe is converted into an inert state. To ensure that the ends of the padlock probe cannot ligate to each other during a ligation reaction with the silencing oligonucleotide as template, the padlock probe binding regions of the silencing oligonucleotide are separated by a gap, which allows the padlock probe ends to separate from each other when hybridizing to the silencing oligonucleotide.

[0010] Therefore, the first aspect provided herein is a method for detecting a target nucleic acid molecule in a sample, the method comprising:

[0011] (i) contacting the sample with a padlock probe comprising target binding regions at its 5' and 3' ends that are complementary to the probe binding site in the target nucleic acid molecule;

[0012] (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 from each other by at least 5 nucleotides, and wherein the contacting occurs before, during or after the sample is contacted with the padlock probe;

[0013] (iii) hybridizing and juxtaposing the target-specific binding region of the padlock probe and the target nucleic acid molecule so as to directly or indirectly link to each other;

[0014] (iv) directly or indirectly ligating the 5' and 3' ends of the padlock probe to circularize the padlock probe;

[0015] (v) after the silencing oligonucleotide is contacted with the padlock probe, performing an RCA reaction using the circularized padlock probe as an RCA template to generate an RCA product (RCP);

[0016] (vi) During or after the RCA reaction, RCP is detected to detect the target nucleic acid molecule.

[0017] The silencing oligonucleotide can be considered as a blocking oligonucleotide for the padlock probe. In addition, since the silencing oligonucleotide performs a function equivalent to washing by "blocking" unhybridized or unligated padlock probes, it can also be referred to as a "washing oligonucleotide" (similarly, the silencing region of an oligonucleotide can also be referred to as a "washing region").

[0018] A feature of the method is that the RCA reaction does not occur (i.e. is initiated) until after the step of contacting the silencing oligonucleotide with the padlock probe. In other words, RCA does not start until the silencing oligonucleotide is present in the sample or reaction mixture (and is able to bind to any free padlock probe). More specifically, the RCA reaction is performed (i.e. initiated) after contact with the silencing oligonucleotide and after ligation of the padlock probe hybridized to the target. More specifically, the RCA reaction is initiated only after said contact or after said contact and ligation, although it may be a very short time later.

[0019] The RCA reaction is usually initiated by adding one or more reagents for RCA (referred to as "RCA reagents"). Typically, the RCA reagents are a polymerase and nucleotides (particularly dNTPs) for RCA, although RCA primers may also be added selectively. One or more RCA reagents may be added earlier, as long as not all RCA reagents are present earlier. In one embodiment, the RCA reaction is initiated by adding at least a polymerase.

[0020] The method is particularly suitable for homogeneous, in-solution formats. Thus, in one embodiment, the method is performed in solution, i.e. in the absence of a solid phase, at least until step (v). In other words, in one embodiment, the padlock probe contacting step, the silencing oligonucleotide contacting step, the padlock probe hybridization and ligation step and the RCA step are all performed in solution, or in the absence of a solid phase.

[0021] In an advantageous embodiment, the method is carried out in a single reaction vessel (eg a single test tube).

[0022] As will be discussed in more detail below, a target nucleic acid molecule can be the analyte, or it can be a nucleic acid molecule that is used or generated as a surrogate, reporter, or indicator of an analyte to be detected.

[0023] In one embodiment, the silencing oligonucleotide is pre-hybridized, pre-mixed, or added to the sample together with the padlock probe. In other embodiments, the silencing oligonucleotide is contacted with the sample after the padlock probe is hybridized to the target nucleic acid molecule or after the padlock probe has been ligated.

[0024] In one embodiment, 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.

[0025] In another aspect, the present invention also provides a kit for detecting a target nucleic acid molecule in a sample, the kit comprising:

[0026] (i) a padlock probe comprising target binding regions at its 5' and 3' ends that are complementary to the probe binding site in the target nucleic acid molecule; and

[0027] (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.

[0028] As described above, the silencing oligonucleotide may optionally comprise a third silencing region. Thus, the fragment of nucleotides (i.e., the nucleotide sequence) separating the first and second silencing regions may or may not hybridize with the backbone portion of the padlock probe. The backbone is the portion between the target binding regions in the padlock probe.

[0029] In this aspect, the padlock probe and the silencing oligonucleotide may be as described above.

[0030] In another aspect, the present invention also provides a silencing oligonucleotide for blocking the target binding region of a padlock probe, wherein the silencing oligonucleotide comprises a first and a second silencing region 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, and wherein the silencing oligonucleotide further comprises a third silencing region, which is located between the first and second silencing regions and is complementary to the silencing oligonucleotide binding site in the padlock probe located between the target binding regions of the padlock probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A reaction scheme for detecting a target nucleic acid molecule using a padlock probe is shown. The target binding regions of the probe 5' and 3' ends are adjacent to each other and are depicted as directly hybridizing on the target nucleic acid molecule. The two ends are ligated together using a ligase to form a circular template, which is amplified by RCA using Phi29 polymerase to generate long linear concatemer RCPs. The RCPs are detected using a labeled detection oligonucleotide.

[0032] Figure 2 Hybridization of a padlock probe to a silencing oligonucleotide is shown; (A) a silencing oligonucleotide having two silencing regions, each region complementary to one end of the padlock probe, separated by a sequence that is not complementary to the padlock probe, thereby forming a gap between the hybridized ends of the probe; (B) and (C) alternative configurations of the padlock probe hybridizing to a silencing oligonucleotide having a third silencing region, wherein the third silencing region hybridizes to the middle portion ("backbone" or backpiece) of the padlock probe located between the two terminal regions.

[0033] Figure 3 The results of the model system experiment as described in Example 1 are shown. A synthetic template containing 9 different target sequences is added to a group of test tubes (referred to as "positive samples"). A control group test tube (referred to as "negative sample") does not contain the synthetic template, but contains a non-target synthetic template molecule (referred to as "wrong template"). A ligation mixture containing 9 padlock probes targeting 9 target sequences, 9 related silencing oligonucleotides (one for each padlock probe) and a ligase is added to the test tube and incubated. An RCA mixture containing RCA reagents and detection beads is added and an RCA reaction is performed. The upper figure shows the results of 8 repeated detections of the same positive sample, and the lower figure shows the results of 8 repeated detections of the same negative sample.

[0034] Figure 4 Shown are (A) experimental results using the model system of Example 1, comparing the correct target molecule (synthetic template) with the incorrect template molecule; (B) titration of silencing oligonucleotides in the model system, 5 nM padlock probe, and 5, 4.5, 4, 3.5, 3, 2.5, 2, and 1.5 nM silencing oligonucleotides.

[0035] Figure 5 Results of clinical samples are shown. Samples in tubes 1-20 were confirmed positive for COVID-19 by PCR, and samples in tubes 21-40 were confirmed negative for COVID-19 by PCR. (A) shows the results in the absence of silencing oligonucleotides, and (B) shows the results in the presence of silencing oligonucleotides.

[0036] Figure 6Results from a model system are shown, demonstrating that the use of a triple-binding silencing oligonucleotide can improve sensitivity. (A) Titration results of the template oligonucleotide in the model system in the absence of the silencing oligonucleotide. (A) Results are shown for the template oligonucleotide at 5nM, 500pM, 50pM, 5pM, 500fM, 50fM, 5fM, and a negative control (Neg C), and the binding of different detection oligonucleotides to the rolling circle product (RCP) or free padlock probe. In this experiment, the free padlock probe was not inhibited from interacting with the RCP by the silencing oligonucleotide. The detection limit was between 5pM and 50pM. (B) Titration results of the triple-binding silencing oligonucleotide and the template oligonucleotide in the model system. (B) Results are shown for the template oligonucleotide at 5nM, 500pM, 50pM, 5pM, 500fM, 50fM, 5fM, and a negative control (Neg C), and the concentration of the silencing oligonucleotide to the padlock probe was 1:1. The results showed that the silencing oligonucleotide allowed the RCP to grow without interference from the free padlock probe that bound to the growing RCP with one arm. If the padlock probe is allowed to bind to one of its arms, the RCP will become double-stranded. Only perfect binding of the padlock probe to the growing RCP is possible (because the padlock probe prefers to bind to the perfect target (18+18nt(36nt)) rather than the triply bound silencing oligonucleotide; however, if the free padlock probe binds to the RCP with one arm, the silencing oligonucleotide will bind to the other arm and prevent the phi29 polymerase from making the product double-stranded), which allows the generation of the second generation RCP. The silencing oligonucleotide allows the padlock probe to bind perfectly to the template, thereby increasing the sensitivity of the detection to a detection limit as low as 5fM to 50fM.

[0037] Figure 7Data comparing two silencing oligonucleotide designs in a model system are presented, at different template oligonucleotide concentrations of 5nM, 500pM, 50pM, 5pM, 500fM, 50fM, 5fM, as well as a negative control (Neg C), and at a 1:1 ratio of concentration to the padlock probe. The "double binding" silencing oligonucleotide in (A) has two silencing regions (binding to two sites), while the "triplet binding" silencing oligonucleotide in (B) has three silencing regions (binding to three sites). The results show that the silencing oligonucleotide with three silencing regions enabled a second round of RCA to proceed (on top of the first round of RCP). This only occurred when the silencing oligonucleotide bound to the padlock probe at three sites (B). The silencing oligonucleotide that does not bind to the padlock probe backpiece (A) does not achieve this lower sensitivity - it does not enable a second round of RCA to proceed. The silencing oligonucleotide with three binding sites allows perfect binding of the padlock probe to the template, thereby increasing the sensitivity of the assay to a detection limit between 50 fM and 5 fM, compared to a detection limit of 50 pM to 5 pM when two binding sites are used.

[0038] Figure 8 Two different silencing oligonucleotide designs with three silencing regions (padlock probe binding sites) are shown: (A) "Danish" configuration (BD) and (B) "Omega" configuration (BO). The first and second silencing regions that bind to the target binding regions at the ends of the padlock probe are called "arms", while the third silencing region that binds to the middle part (backbone) of the padlock probe is called Bp ("backbone"). DETAILED DESCRIPTION

[0039] 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 a target nucleotide sequence (the term "nucleotide sequence" can be used interchangeably with "nucleic acid sequence").

[0040] The method is based on detecting a target nucleic acid using a padlock probe, which is ligated and subjected to a rolling circle amplification (RCA) reaction after binding to its target. The padlock probe is detected by detecting the product of the RCA reaction, thereby detecting the target nucleic acid. The improvement relates to an improved method for removing or clearing free padlock probes that are not hybridized and not ligated. The free padlock probe is cleared by blocking with a silencing oligonucleotide, making it unable to participate in any non-specific or unnecessary reactions that may interfere with detection and / or produce non-specific signals. In this way, the specificity and / or sensitivity of the detection method described herein can be improved. Therefore, "removal" or "clearance" as described herein does not require physical separation from the sample, but includes functional removal or clearing. When the padlock probe is silenced or blocked by the silencing oligonucleotide, it cannot be connected, or is substantially unable to be used for connection. In addition, it cannot or is substantially unable to trigger an extension reaction on any other nucleic acid template that may be present or available in the sample, because when it is hybridized with the silencing oligonucleotide, it cannot or is substantially unable to hybridize with any other nucleic acid molecule that may serve as an RCA template.

[0041] As will be described in more detail below, in the detection method based on padlock probe-RCA, RCA product (RCP) can be conveniently detected by hybridizing the detection oligonucleotide to RCP, and these detection oligonucleotides are detected in RCP. In order to enable the detection oligonucleotide to be attached to RCP, RCP must maintain its single-stranded form generated by RCA by circularized padlock probe. Therefore, it is important to prevent the use of RCP as an extension template when unwanted extension reaction occurs, otherwise RCP will be made double-stranded. Therefore, it is not desirable that the free padlock probe that is not hybridized with the target nucleic acid molecule or the padlock probe that is not connected in the previous step can hybridize with RCP and trigger an extension reaction.

[0042] Thus, in one embodiment, the purpose of the silencing oligonucleotide is to effectively bind to any free unbound or unligated padlock probe present in the reaction mixture (the mixture being produced by contacting the sample with the padlock probe and allowing the probe to hybridize or hybridize and ligate), thereby isolating it. Thus, in one embodiment, the role of the silencing oligonucleotide is to prevent, or reduce or inhibit, any free padlock probe (i.e., a padlock probe that is not hybridized to the target nucleic acid or unligated) present in the reaction mixture from hybridizing to or being able to hybridize to the RCP.

[0043] Thus, the silencing oligonucleotide is contacted with the padlock probe before the RCA reaction is performed (ie before the RCA reaction is initiated).

[0044] However, although it is generally undesirable for unreacted padlock probes to bind to RCPs in an RCA reaction, in a so-called super RCA (sRCA) reaction, 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, thereby generating a second-generation RCA product by RCA of the padlock probe attached to the first RCP (to produce a so-called "second-generation RCP"). We have found that certain designed silencing oligonucleotides, i.e., silencing oligonucleotides having three silencing regions, can promote or enable this second-round RCA reaction (or sRCA). This will be described in more detail below.

[0045] In certain embodiments, the silencing oligonucleotide / padlock probe pairing can be designed to minimize the possible displacement of the silencing oligonucleotide from the hybrid of the silencing oligonucleotide and the padlock probe after RCP generation. In addition, by adding detection oligonucleotides to the RCA reaction mixture, these detection oligonucleotides can hybridize with the RCP when it is generated, thereby minimizing the risk of any such possible displacement. In addition, the silencing oligonucleotide / padlock probe hybrid itself can hybridize with the RCP through another part of the padlock probe. This combined padlock probe will not be able to be amplified by RCA.

[0046] In other embodiments, the silencing oligonucleotide / padlock probe pair can be designed to preferentially bind the padlock probe to its target (or "template") sequence, such that in the presence of the target sequence, the padlock probe will bind to the target sequence rather than the silencing oligonucleotide. This situation is suitable for sRCA reactions where a second round of RCA is required.

[0047] By "removing" or blocking unreacted padlock probes in this way, the method is particularly suitable for homogeneous detection, that is, detection performed in solution and without the need for a solid phase. However, its use in heterogeneous or solid-phase based methods is not excluded.

[0048] As mentioned above, the use of padlock probes and RCA for detecting a target nucleic acid according to the methods described herein is known in the art. Figure 1The operation of this detection method is schematically shown. Padlock probe and target nucleic acid hybridization. As will be discussed in more detail below, according to the conformation of padlock probe, the target binding region of padlock probe may be able to hybridize with the target sequence in the target molecule that it wishes to detect (or in other words, be complementary to it). Or, the target binding region of padlock probe may be able to hybridize with the binding site located at both sides of the target sequence in the target nucleic acid molecule (or be complementary to it). The latter situation may be applicable to, for example, when padlock probe is a gap filling type padlock probe, wherein the gap between the 5' and 3' ends of padlock probe hybridization is extended and filled by the hybridization 3' end catalyzed by padlock probe polymerase, using target nucleic acid molecule as extension template. Padlock probe is contacted with the target nucleic acid molecule in the sample, and it is hybridized with the target molecule.

[0049] The term "contacting" as used herein broadly includes any method of contacting a sample (or more specifically, a target nucleic acid molecule) with a padlock probe. This may involve, for example, adding a padlock probe to a sample and maintaining (e.g., incubating) a reaction mixture under conditions that allow the probe to hybridize with the target nucleic acid molecule. Alternatively, a sample or an aliquot thereof or a portion thereof may be added to a padlock probe or a reaction mixture comprising a padlock probe.

[0050] When the padlock probe hybridizes to the target nucleic acid, the ends of the padlock probe are juxtaposed so as to ligate together: directly if the ends hybridize directly adjacent to each other, or indirectly if a gap exists between the ends of the padlock probe when hybridizing. As described above and in more detail below, the gap can be filled by a gap oligonucleotide or a gap-filling extension. Figure 1 As shown, the ends of the padlock probes are directly hybridized adjacent to each other and can therefore bind directly by ligation. By ligation, the padlock probes are circularized, thereby generating a circular template for the RCA reaction. The RCA reaction generates a long concatemer product (RCP) containing multiple repeats of complementary copies of the circularized padlock probes, connected in series. In other words, the RCP contains multiple monomers, connected to each other, each monomer representing a complementary copy of the circularized padlock probe. As shown Figure 1 As shown, the padlock probe contains a detection sequence that is copied into each monomer repeat in the RCP and provides a binding site for the detection oligonucleotide. The hybridization of the detection oligonucleotide with its multiple binding sites causes it to be concentrated in the RCP, thereby enabling it to be detected with high sensitivity. As described in more detail below, the detection oligonucleotide can carry a detectable portion (e.g., a label) to enable it to be detected. Figure 1 As shown, the labels are colored magnetic beads, but any detectable label may be used.

[0051] Any padlock probe that fails to hybridize to the target or fails to bind may or will be free in the sample, or more specifically in the reaction mixture. In order to prevent these unbound or unbound padlock probes from being able or having the opportunity to hybridize to the generated RCP and trigger an unwanted extension reaction, a silencing oligonucleotide is used to "capture" or "block" these probes before the RCA reaction is initiated. More specifically, the target binding region of the padlock probe is blocked by binding to the silencing oligonucleotide, for example Figure 2 shown.

[0052] In general, the method is used to detect target nucleic acid molecules. More specifically, the method is used to detect target nucleic acid sequences in target nucleic acid molecules. The term "detection" used herein broadly includes any method for determining the presence of target nucleic acid molecules. In the present method, the target nucleic acid is detected by detecting the presence or quantity of the generated RCP, which may include simply detecting whether it exists, or any form of measurement of the RCP. Therefore, the RCA product generated in step (v) can be detected as a "signal" of the target nucleic acid molecule. Therefore, detecting RCP in step (vi) includes determining, measuring, evaluating or analyzing the presence, absence, amount or position of RCP in any way. The presence of RCP (i.e., confirming its presence or amount) indicates or identifies the presence of the target nucleic acid molecule, because the successful generation of RCP depends on the presence of the target nucleic acid molecule (or more specifically, the target nucleic acid sequence).

[0053] Quantitative and qualitative determination, measurement or assessment are all included, including semi-quantitative. These determinations, measurements or assessments can be relative, such as when detecting two or more different target nucleic acid sequences or target molecules in a sample, or can be absolute. Therefore, in one embodiment, the method can be used to quantify or determine the amount of the target nucleic acid molecules or sequences present. The term "quantification" can refer to absolute quantification or relative quantification in the context of the target nucleic acid molecules or sequences used to quantify the sample. Absolute quantification can be achieved by comprising one or more control nucleic acid molecules of known concentration and / or by referencing the level of the detected target nucleic acid molecules or sequences to known control nucleic acid molecules or sequences (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection level or amount between two or more different target nucleic acid molecules or different target sequences, to provide the relative quantification of each of two or more different nucleic acid molecules or sequences, i.e., relative to each other. Therefore, as described above, the ratio of the target nucleic acid molecules or sequences in the sample can be determined. For example, the copy number of the target nucleic acid molecules (such as chromosomes) can be compared.

[0054] The target nucleic acid molecule is any nucleic acid molecule that is desired to be detected, in other words, it is the target of detection. More specifically, the target can be a target sequence present in a nucleic acid molecule. In certain 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, can be detected. The method can be carried out in a multiple manner to detect, for example, two or more different target sequences in one or more target nucleic acid molecules, and / or target sequences in two or more target molecules. Therefore, in order to detect the target sequence in two or more different target molecules, a variety of first padlock probes can be used, each probe corresponding to different target sequences, i.e., having a target binding region complementary to the binding site in the target sequence, or, for example, being located at the flank of different target sequences. In this respect, it should be understood that if the target binding site is located at the flank of the target sequence in the molecule, the flank binding sites in different target molecules may vary due to different target sequences, to achieve the specific binding of the padlock probe. Therefore, in this case, the flank sequence targeted by the padlock probe is distinguishable between different targets. Therefore, they can be considered as part of the target sequence. Alternatively, or in addition, different and separate target sequences in the same target molecule can be detected, for example different sequences in different genes on a chromosome, again using a plurality of different padlock probes, each corresponding to a different target sequence.

[0055] In a specific embodiment, the method can be used to detect which of a plurality of possible different variant sequences is present in a given target molecule, such as whether a wild-type or mutant sequence is present, or which of a plurality of possible mutations, different allelic variations, or polymorphisms. In this scheme, the padlock probe can include a target binding region designed to distinguish between different variations. Alternatively, a gap-filling padlock probe can be used, which hybridizes to the flanking regions flanking all or multiple variations, and performs a gap-filling extension step that generates a complementary copy of the variant sequence present. By detecting the complementary sequence of the variant sequence present in the RCP, the nature of the variant sequence can be determined or the variation can be detected. For example, this can be achieved by sequencing or using a detection oligonucleotide specific to the variant sequence.

[0056] Therefore, the method can be used for detecting multiple target molecules or target sequences.Term " multiple (multiple) " used in this article means two or more, for example 3,4,5,6,8,9,10,15,20,25,30,40,50,60,70,80,90 or 100 or more.In fact, thousands or even tens of thousands of padlock probes can be used.The number of padlock probes that can be used is not limited, and can be adjusted as needed.This will depend on the purpose of the method, the nature of the sample, the target nucleic acid sequence to be detected, the possible number of variations, etc.Therefore, for example, in order to detect wild-type and mutant-type variation, the number of different padlock probes will depend on the possible number of mutations, for example 2-6,2-5,2-4 or 2-3 different padlock probes.It should be understood that different aspects can be combined to increase the overall multiplicity of detection.For example, in a given sample, the method can be used for detecting the different variations of different target sequences.

[0057] The target nucleic acid molecule or sequence can be any molecule or sequence that is desired to be detected or identified. It can be DNA or RNA, or a modified variant thereof. Therefore, the nucleic acid can be composed of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. Therefore, the nucleic acid can be or can include, for example, bisulfate-converted DNA, LNA, PNA, or any other derivative containing a non-nucleotide backbone.

[0058] Typically, the target molecule or sequence will be the analyte that you want to detect, such as a nucleic acid present in a sample, such as a cell or tissue sample or any biological or clinical sample, etc. It may be a viral nucleic acid. Therefore, it may be a naturally occurring sequence, or a derivative, copy or amplicon thereof. However, the method is not limited to this, and the target molecule may also be a reporter molecule for detecting an analyte. In the process of detecting any analyte (such as a protein or other biomolecule, or a small molecule in a sample), a reporter nucleic acid can be used or generated. Therefore, the reporter nucleic acid can be used as a label or marker for the analyte binding probe, and the analyte can be detected by detecting the reporter nucleic acid, for example in an immunoassay, such as an immunoPCR or immunoRCA reaction. The reporter nucleic acid can be generated during the detection process, such as by a ligation reaction in a proximity ligation assay, or an extension reaction in a proximity extension assay, or by a cleavage reaction, etc. Therefore, this reporter target nucleic acid can be a synthetic or artificial sequence.

[0059] In one embodiment, the target nucleic acid is a DNA molecule, natural or synthetic. The target nucleic acid molecule can be coding DNA or non-coding DNA, such as genomic DNA or a subcomponent thereof, or can be derived from genomic DNA, such as a copy or an amplicon thereof, or it can be cDNA or a subcomponent thereof, or an amplicon or a copy thereof, etc.

[0060] 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 an RNA pool, such as a genomic nucleic acid, whether human or from any source, from a transcriptome or any other nucleic acid (e.g., an organelle nucleic acid, i.e., a mitochondrial or plastid nucleic acid or a viral nucleic acid), whether naturally occurring or synthetic. Therefore, the target RNA molecule may be or may be derived from a coding RNA (i.e., pre-mRNA or mRNA) or a 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 a 16S RNA, such as a microorganism (e.g., a pathogenic microorganism) from a sample and used to identify the microorganism. Alternatively, the target RNA molecule may be a genomic RNA, such as a single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) of a virus with RNA as genetic material. Notable examples of 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, negative-sense, or double-stranded RNA from a viral genome, or positive-sense RNA from a retroviral RNA genome.

[0061] If the target molecule is an RNA molecule, the method may comprise a 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).

[0062] Alternatively, the target RNA molecule can be contacted with the padlock probe directly. In other words, the first padlock probe can bind directly to the target RNA molecule.

[0063] As mentioned above, in order to detect the variant target sequence, the padlock probe may have specificity to a particular variation. However, as also mentioned above, this is not necessary, and in another embodiment, the padlock probe that can capture any possible variation of a given target sequence can be used. Therefore, this padlock probe does not have selectivity or specificity to any particular variant. However, in order to ensure that the required target sequence is captured, the padlock probe will be designed to specifically bind to the target molecule on the site that makes all target sequence variations captured, i.e., be positioned at the target sequence flank and a site shared between different variants. For example, the variant sequence may be a mutation or a polymorphism in a specific position or site in a gene. Therefore, the target sequence may be a sequence comprising the variant position or site, and different target sequences can be distinguished by having different bases in this position or site. The padlock probe can be designed to have a binding site complementary to the flanking sequence shared or conservative (i.e., common) between different variants in the target molecule.

[0064] Therefore, in this embodiment, the padlock probe can be considered as a universal probe, or has universality to a group of target sequences or a group of variant target sequences. Therefore, the padlock probe may have a target binding region that is complementary to a binding site (i.e., a flanking region) in a target molecule, and the binding site is common to different target sequences (i.e., a common binding site flanking different target sequences or different variants of a target sequence). In other words, the padlock probe may have a target binding region that is common or universal to different target sequences or different variants, or is common to a group of target sequences or a group of variants. In other words, the padlock probe may have a target binding region that is capable of hybridizing to a complementary binding site common to different target sequences or different variants of a target sequence in a target molecule.

[0065] In another embodiment, the padlock probe may be specific for a particular target nucleic acid sequence. Thus, for example, different padlock probes may be used, each probe being specific for a different target sequence. This may be useful for diagnostic tests, such as NIPT, where different sequences may be detected, such as detecting different chromosomes (and determining their copy number, such as detecting trisomy), or in any situation where it is desired to detect one or more specific target sequences.

[0066] Padlock probes can also be defined as circularizable probes. The use of padlock or circularizable probes is well known in the art, including in the context of RCA reactions. Circularizable probes include one or more linear oligonucleotides that can be linked together to form a ring. Padlock probes are well known and widely used, and are reported and described in detail in the literature. Therefore, the principle of padlock probes is well understood, and the design and use of padlock probes are known and described in the art. Padlock probes are generally a linear cyclizable oligonucleotide that is hybridized with its target nucleic acid sequence or molecule in a certain way so that the 5' and 3' connectable ends of the probe are juxtaposed to connect together, whether directly connected or indirectly connected as described above, with a gap in between. By connecting the hybridized 5' end and 3' end of the probe, the probe is cyclized. It is understood that in order to allow cyclization (connection) to occur, the connectable 5' end of the padlock probe has a free 5' phosphate group.

[0067] In order to juxtapose the ends of the padlock probe for ligation, the padlock probe is designed to have target binding sites at its 5' and 3' ends. That is, the complementary regions that allow the padlock probe to bind to its target are located at the ends of the padlock probe.

[0068] In order to connect them, the 3' end and 5' end to be connected (the "connectable" 3' end and 5' end) are hybridized with the target molecule or sequence, which serves as a connection template. The combination of the padlock probe puts the ends in the described juxtaposition. If the complementary binding sites in the target molecule or sequence are directly adjacent to each other (or connected), the ends of the padlock probe will hybridize directly adjacent to each other (i.e., there is no gap) and can be directly connected to each other. Therefore, in this case, the connectable end of the probe is provided by the actual end of the probe. In the case of a gap-filling padlock probe, the target binding region at the end of the padlock probe does not hybridize with the adjacent binding site, but hybridizes with the non-adjacent (non-connected) binding site in the target molecule. In this configuration, the connectable 5' end of the probe is provided by the actual 5' end of the probe. However, the connectable 3' end of the probe is generated by extending 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' end of the probe for connection. Thus in this case the ligatable 3' end of the probe is the extended 3' end of the probe.

[0069] The padlock probe can be provided in two or more parts, which are connected together. This may involve providing an additional connection template, such as in the case of a two-part probe, wherein each part only comprises a target binding region, and the other end of each part is hybridized with a common connection template. In another embodiment, the two-part padlock can take the form of a "connector" or "backbone" oligonucleotide, respectively at or near the 5' and 3' ends, which have two target binding regions, which are hybridized with the target, with a gap in the middle, and with a gap oligonucleotide hybridized in the gap between the ends. The gap oligonucleotide can partially or completely fill the gap. In the case of a gap-filling padlock probe, the 3' end of extension can be the hybridization 3' end of a gap oligonucleotide or a backbone oligonucleotide.

[0070] However, in typical embodiments, the pad is provided as a single circularizable oligonucleotide, whether a gap-filling pad or a non-gap-filling pad.

[0071] In some specific embodiments, the padlock probe does not have a secondary structure, more specifically, does not include an intramolecular double-stranded region or a stem-loop structure. However, a dumbbell probe does have a secondary structure, which is a specific subtype of a padlock probe. The dumbbell probe comprises two stem-loop structures, and the stem is connected to the stem, wherein one "loop" is not closed, but open, with connectable 5' and 3' ends. This "open loop" serves as the target binding domain of the probe. The closed loop serves only as a spacer connecting the ends of the duplex (stem). In other words, it can be regarded as a padlock probe with a duplex region formed between the complementary sequences (regions) of the padlock. The duplex region serves as a signal domain, and an intercalator can be combined with it. Therefore, the "open loop" of the dumbbell probe can include a complementary target binding region.

[0072] The padlock probe may comprise one or more other regions or sequences that may be useful in the method. Specifically, this may include a detection sequence that allows the probe to be detected or identified. The detection sequence may be, for example, an identification sequence, such as a barcode sequence, which can be used to detect the padlock probe (more specifically, the complementary sequence of the detection sequence in the RCP can be detected) by detecting the sequence. Alternatively, the detection sequence, more specifically its complementary sequence, can be detected by a detection oligonucleotide that hybridizes with the complementary sequence of the detection sequence in the RCP. Therefore, the detection oligonucleotide is a detection probe that detects RCP by detecting it, thereby detecting the padlock probe for generating the RCP, and then detecting the target nucleic acid molecule. The detection oligonucleotide may be equipped with a detection portion, as discussed further below. A complementary copy of the detection sequence in the padlock probe is generated in each monomer repeat in the RCP. This complementary copy in the RCP can hybridize with the detection oligonucleotide, so it itself can be complementary to the detection oligonucleotide. Therefore, it can be seen that the detection sequence in the padlock probe can correspond to the sequence of the detection oligonucleotide for hybridizing with the RCP.

[0073] In the method, the target sequence to be detected can include one or more variant bases. Therefore, it can be a single nucleotide variation, such as a single nucleotide polymorphism (SNP) or a sudden change, or it can include two or more bases. Therefore, the variant sequence can include a section of nucleotides, wherein two or more bases may be variants. The variant base can be continuous or non-continuous.

[0074] The length of the target sequence is not critical and can vary depending on the circumstances, the nature of the target molecule, the target sequence or the position or site of the variation. Thus, the target sequence can be, as a representative example only, 1 to 10 nucleotides, such as 1-15, 1-12, 1-10, 1-8, 1-7 or 1-6 nucleotides long. However, in certain embodiments, a target sequence longer than a single nucleotide may be beneficial to improve specificity, in which case the target sequence can be any one of 2, 3, 4, 5 or 6 to any one of 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides long. Thus, exemplary target sequences can be 4-10, 4-8, 4-7, 4-6, 5-10, 5-8, 5-7 or 6-8 nucleotides long.

[0075] The silencing oligonucleotide is a linear oligonucleotide comprising at least two silencing regions, i.e. at least a first and a second silencing region. The only requirement is that the silencing region is able to hybridize to the target binding region at the end of the padlock probe, e.g. Figure 2 As shown, when hybridized to the silencing oligonucleotide, there is a gap of at least 5 nucleotides between the ends of the padlock probe. Thus, the silencing regions are separated by gap sequences in the silencing oligonucleotide.

[0076] In particular, the gap sequences between the silencing regions may be at least 6, 7, 8, 9 or 10 nucleotides long.

[0077] For example, the gap sequences between silent regions can be 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 5-40, 6-40, 7-40, 8-40, 9-40, 10-40, 5-30, 7-30, 8-30, 10-30, 5-25, 7-25, 8-25, 10-25, 5-20, 7-20, 8-20, or 10-20 nucleotides in length.

[0078] In one embodiment, the silencing region can be located at both the 5' and 3' ends of the silencing oligonucleotide, but this is not required.

[0079] The silencing oligonucleotide may comprise further silencing regions which are complementary to other sequences in the padlock probe located between its ends, i.e. to sequences within the padlock probe sequence. In particular, the silencing oligonucleotide may comprise a third silencing region. This Figure 2 B and C and Figure 8 The third silencing region is located between the first and second silencing regions in the silencing oligonucleotide. It is complementary to the silencing oligonucleotide binding site in the padlock probe, which is typically located between the 5' end and the 3' end of the padlock probe. Such silencing oligonucleotides are referred to herein as "triplet binding" or "triplet binding bodies" and represent a particularly advantageous aspect.

[0080] For example, when the third silencing region hybridizes to the padlock probe, the hybridized 3' end of the padlock probe may be prevented from being extended by a polymerase used in an RCA reaction.

[0081] Therefore, it is understood that the third or further silent region is located in the gap sequence. It may correspond to the entire gap sequence, or a part of the gap sequence. Therefore, the gap sequence can be or can include a sequence complementary to the sequence of the non-target binding region in the padlock probe. In this configuration, the third silent region can be partially complementary to any non-target binding region in the padlock probe. This silent oligonucleotide binding site is generally located between the target binding region at the 5' end and the 3' end of the padlock probe, or between the backbone portion of the two-part padlock probe. The third silent region can be referred to as being combined with the backbone portion of the padlock probe, or as a backbone silent region.

[0082] In one embodiment, the third silencing region may be capable of hybridizing to (e.g., may be complementary to) all or part of the detection sequence of the padlock probe. In other words, the detection sequence may fully or partially overlap with a silencing oligonucleotide binding site that is complementary to the third silencing region of the silencing oligonucleotide.

[0083] Alternatively, if the silencing oligonucleotide does not comprise any further silencing region in addition to the first and second silencing regions, the gap sequence is not complementary to any part of the padlock probe.The gap sequence may be, for example, a random sequence.

[0084] As described above, the triplicated conformation of the silencing oligonucleotide allows the RCP generated in the first round (i.e., the "first RCP") to serve as a ligation template for the padlock probe for the second round of RCA. Figure 7 In the embodiment of the present invention, it has been shown, and described in the embodiment below.It has been found that three combinations of silent oligonucleotides (respectively attached to the first and second target binding regions of the padlock probe, also referred to herein as "arms" of the padlock probe) with the first and second silent regions and the third silent region attached to the padlock backbone may make the padlock probe less inclined to attach silent oligonucleotides than to attach its target sequence.In other words, in this conformation, the padlock probe may be more inclined to attach its target sequence rather than silent oligonucleotides.In the absence of a target sequence, or when the padlock binding site in the target sequence is blocked or occupied, the silent oligonucleotide will attach to the padlock probe.However, in the presence of a target sequence (also referred to as "template sequence"), the padlock probe will attach to the target sequence.Although it is not desired to be bound by theory, it is assumed that this may be due to the padlock probe-target sequence binding structure being less rigid than the padlock probe-silent oligonucleotide binding structure.

[0085] The target binding site (arm) of the padlock probe is complementary to the target sequence. The first RCP generated by the RCA of the connected padlock probe contains a monomer complementary to the padlock probe, so it can be seen that the first RCP contains repeated homologous copies of the target sequence in its monomer repeats. Therefore, these repeated copies are copies of the target sequence and provide binding sites for the padlock probe. In the presence of a ligase, studies have shown that since the padlock probe preferentially binds to the target sequence, the padlock probe can be connected and used as a template for a second round of RCA to generate a second generation RCA product ("second RCP"). This leads to signal amplification. The first RCP contains repeated binding sites for multiple padlock probes, so multiple second RCPs can be generated. This sRCA reaction is known in the art.

[0086] A person skilled in the art can easily design a cognate silencing oligonucleotide / padlock probe pair for use in an RCA or sRCA reaction as desired. It is known how to design oligonucleotides with different hybridization strengths to achieve preferential hybridization to a desired hybridization binding pair.

[0087] The length of the silent region of the silent region is not critical, as long as they are long enough to achieve specific hybridization with the complementary binding site in the padlock probe. Equally, they can change according to the design and the selected conformation combined with the padlock probe. The first and second silent regions are bound to the target binding site of the padlock probe. Although they do not need to correspond to the target binding site completely, they must be long enough to specifically bind these regions. The first and second silent regions are usually at least 6 nucleotides long. The third silent region can be shorter, for example at least 2 nucleotides long. As described in the following embodiments, various lengths have been studied and found to be effective.

[0088] In representative embodiments, the first and second silencing regions can be at least 6, 7, 8, 9 or 10 nucleotides long. The maximum length is not critical, depending on the length of the target binding site of the padlock probe. For example, they can be up to 7, 8, 9, 10, 11, 12, 15, 20, 25 or 30 nucleotides long, or longer (if necessary). Representative ranges include any range between the above minimum or maximum integers, such as 6-30, 6-20 or 6-18. Other ranges include 6-15, 6-12, 7-20, 8-20, 9-20, 10-20, etc. The third silencing region can be, for example, 2-50, 2-40 or 2-30 nucleotides long.

[0089] Different combinations of first / second and third silencer region lengths can be used. For example, first and second silencer regions of 18 nt can be used in combination with a third silencer region of 20 nt and have been shown to work well, but this can be varied to be used in combination with a shorter third silencer region. Similarly, first and second silencer regions of 10 nt can be used in combination with a third silencer region of 20 nt and work well.

[0090] As described above, the first and second silencing regions need not correspond exactly in length to the target binding region of the probe, and thus the binding site in the padlock probe that is complementary to the silencing region may be shorter or longer than the binding site for the target sequence of the padlock probe. For example, the first or second silencing region of 10-12 nt in length may be shorter than the target binding site of the padlock probe and may leave a stretch of nucleotides at the end of the padlock probe free (i.e., the nucleotides most 3' and / or 5' of the probe, i.e., the nucleotides closest to the ligation site).

[0091] One silencing oligonucleotide is provided for each padlock probe used. In other words, there is one cognate silencing oligonucleotide for each padlock probe. Thus, for example, when different target-specific padlock probes are used, each comprising a different target binding region, each padlock probe will have a different cognate silencing oligonucleotide. However, in the case of a single or "universal" padlock probe, only one cognate silencing oligonucleotide is required.

[0092] In order to carry out the method, the sample, or more specifically, the target nucleic acid, is contacted with the padlock probe. This contact may include other reagents. The sample containing the target nucleic acid molecule or its part, component or aliquot can be contacted with the padlock probe. For example, for in situ analysis, or in fact for other analysis, for example for the analysis of clinical samples, the sample can be directly contacted with the reagent. The sample can be pre-treated or processed before contact, for example by fixed cells or tissue samples. In other embodiments, the target nucleic acid can be first separated or removed from the sample. The procedure of extracting or purifying nucleic acid (for example DNA) from various types of samples is well known in the art. For example, nucleic acid can be isolated from cells or cell-free samples (such as blood plasma). In some cases, it may also be necessary to fragment the nucleic acid molecules. The procedure in this regard is known in the art, including specific digestion such as using nucleases, including for example restriction endonucleases, or non-specific methods such as shearing.

[0093] The step of contacting with the padlock probe prepares a reaction mixture for the probe binding step. However, reagents for subsequent steps may also be included, for example, if the padlock probe is a gap filling padlock probe, reagents for the extension step may be included. In addition, in another embodiment, or additionally, reagents for ligation reactions may be included. In general, the initial reaction mixture will not include all reagents required for performing the RCA reaction in order to control the RCA reaction (i.e., start later). However, in certain embodiments, some RCA reagents (e.g., RCA primers or nucleotides) may be included together with the padlock probe, or more generally included in the initial reagent mixture contacted with the sample.

[0094] To perform the probe binding step, the padlock probe is typically incubated with the target nucleic acid molecule. For gap-filling padlock probes, dNTPs and a polymerase may also be included. Conveniently, a ligase may also be included during the probe binding step. Reagents may be added in a single reaction mixture, or separately before or during the probe binding step. To allow the probe to bind, there may be an initial heating step, for example to denature the double-stranded nucleic acid molecule.

[0095] Reagents are typically provided in a buffer according to principles and procedures known in the art. For example, a buffer suitable for the selected ligase can be selected.

[0096] The reaction mixture can be incubated under conditions suitable for promoting or achieving padlock probe binding (a so-called "annealing" step). If there is a denaturation step before, this may involve a reduction in temperature. The conditions of these steps are known in the art and are selected or designed within the routine skills of those skilled in the art. For example, room temperature or an annealing temperature of 20-40° C., such as 25-40° C. or 25-37° C., can be used. In certain embodiments, higher temperatures, such as 50-65° C., such as 53 to 60° C. or 55 to 60° C., can be used.

[0097] If a higher annealing temperature is chosen, the annealing temperature can be lowered during the extension step when using a gap-filling padlock probe. Again, appropriate conditions can be selected based on knowledge in the art and the specific reagents, such as enzymes, used. For example, after the initial annealing step, the temperature can be lowered to 28-40°C, such as 28-35, 30-35, 28-33, 30-33, 28-33, or 30-32°C, etc.

[0098] 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).

[0099] Conveniently, the silent oligonucleotide can contact with the sample while the padlock probe contacts with the sample. In this respect, the silent oligonucleotide can contact alone or in a mixed manner. Therefore, in one embodiment, a reagent mixture comprising the padlock probe and the silent oligonucleotide can be prepared. In another embodiment, the reagent mixture can also include a ligase for the padlock probe ligation step. The mixture can contact with the sample. Alternatively, the reagent can be added to the sample separately. The resulting reaction mixture can then be hatched so that the padlock probe is combined with connection to occur.

[0100] The silencing oligonucleotide may be provided prehybridized to a padlock probe and this reagent contacted with the sample or used to prepare the initial reagent mixture.

[0101] It should be understood in this respect that in this format, the silencing oligonucleotide is pre-hybridized or added to the sample together with the padlock probe, and the target nucleic acid displaces the silencing oligonucleotide from the padlock probe or competes for the silencing oligonucleotide when combined with the padlock probe. In other words, the hybridization of the padlock probe to the target nucleic acid is more advantageous than the hybridization of the silencing oligonucleotide. However, any padlock probe that is not hybridized to the target molecule still remains or will hybridize to the silencing oligonucleotide.

[0102] Alternatively, in another embodiment, a reagent mixture comprising a padlock probe and a ligase can be prepared and contacted with the sample to hybridize the padlock probe to the target nucleic acid. A silencing oligonucleotide can then be added. Any unbound padlock probe present in the reaction mixture is hybridized to the silencing oligonucleotide.

[0103] In another embodiment, the padlock probe can be contacted with the target nucleic acid and allowed to hybridize. A ligase can then be added and allowed to connect any bound padlock probes. Subsequently, a silent oligonucleotide can be added to hybridize with any unhybridized and unconnected padlock probes.

[0104] If the padlock probe is a gap-filling padlock probe, the initial reaction mixture for padlock probe binding may also include a polymerase and nucleotides for the extension step. In such a procedure, a heat inactivation step of the polymerase may be required prior to initiating RCA.

[0105] The padlock probe is ligated to circularize it, thereby generating a template for the first RCA reaction. The ligation is performed using the target nucleic acid molecule as a template. Any convenient ligase may be used, and relevant representative ligases include, but are not limited to, temperature-sensitive ligases such as SplintR ligase (also known as PBCV-1 DNA ligase or Chlorella 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, Pfu ligase and 9°N TM DNA ligase.

[0106] Suitable ligation conditions are known in the art, and any necessary and / or desired reagents can be combined with the reaction mixture and maintained under conditions sufficient for ligation. Obviously, the ligation conditions may depend on the ligase used in the method of the present invention. Thus, for example, Ampligase can be used and the temperature can be increased in the ligation step. Alternatively, SplintR ligase can be used at room temperature.

[0107] If temperature changes or temperature control steps are required, the method can be performed in a thermal cycler instrument. Doing so allows for easy control of temperature changes. However, an advantage of this method is that extreme temperature changes are not required, for example it can be performed at room temperature or 20-37°C. For example, the probe binding and ligation steps can be performed at room temperature.

[0108] The conditions for the probe binding and ligation reactions can be optimized by routine experiments according to principles known in the art. Thus, the temperature, buffer, incubation time, heating rate, etc. can be adjusted to find the optimal conditions.

[0109] Once the padlock probes are attached, the RCA reaction is performed. Prior to the RCA reaction, there may be an optional washing step, for example if the method is performed in a heterogeneous or solid phase format (such as when the sample is fixed on a solid support). However, an advantage of the present method is that no washing step is required. Therefore, it is not preferred to include such a step. In one embodiment, the method proceeds to the RCA step without a washing step.

[0110] The RCA reaction is then started. This can be achieved by adding reagents to the reaction mixture to start the RCA reaction. This can be a complete RCA reaction mixture, which contains all the reagents required for the RCA reaction (e.g., polymerase and nucleotides, and optional primers). Alternatively, one or more reagents can be added. For example, a single reagent can be added. Conveniently, the RCA reaction can be started by adding a polymerase. The polymerase can be conveniently added by adding an RCA reagent mixture containing other reagents for the RCA reaction (e.g., nucleotides) in an appropriate buffer. However, in certain embodiments, other reagents for the RCA reaction can be added or incorporated early.

[0111] As mentioned above, the RCA reaction is well known in the art and thus the conditions for this step can be designed or selected according to protocols and principles known and described in the literature. A strand displacing polymerase is used, such as Phi29 or a derivative thereof.

[0112] The primer of RCA reaction can be added to the reaction mixture, or can be pre-hybridized on the padlock probe. The binding site of RCA primer can be located in the region of the padlock probe that is different from the target binding region (i.e., in the backbone region of the padlock probe). In some cases, the target nucleic acid molecule can be used as a primer or a primer is provided. If necessary, the 3' exonuclease activity of a polymerase or a separate exonuclease can be used to digest the target nucleic acid to provide the hybridization 3' end of the RCA primer suitable for the RCA of the circularized padlock probe.

[0113] The RCA reaction generates concatemer RCA products (RCPs) containing multiple repeated copies of the complementary sequence of the padlock probe. The target nucleic acid molecule is detected by detecting the RCPs.

[0114] The method can be carried out in a heterogeneous or homogeneous format. That is, it can be performed on a solid phase (or support), or in solution or suspension (i.e. without a solid phase or support), or indeed both, since a solid phase can be introduced at a later stage, for example in a step for detecting RCP.

[0115] The format of the method can be selected according to the nature of the sample, the target nucleic acid molecule or the desired readout or detection technology. For example, for liquid or liquefied or dissolved samples, or for isolated or purified nucleic acids, etc., or for detecting non-nucleic acid analytes in samples (e.g., detecting proteins in serum or plasma, or other body fluids, etc.), or indeed for detecting any analyte in a convenient diagnostic or clinical test, a solution phase format can be used.

[0116] On the other hand, in order to detect the target sequence in a solid sample (such as a tissue or cell), for example for in situ detection, a solid phase format can be used. It may also be desirable to fix the sample or target nucleic acid for other reasons, for example for a specific detection format, or simply according to choice. Therefore, the isolated cell sample can be fixed on a support, or nucleic acid can be separated or captured from the sample onto a solid support, or a similar operation is adopted. In another embodiment, the primers for the second RCA reaction can be fixed on a solid support.

[0117] In one embodiment, the method can be used for the localization detection of target nucleic acid molecules, and "localization" detection means that the signal causing the nucleic acid detection is localized to the nucleic acid, in this case, the RCP is localized to the target nucleic acid. Therefore, the nucleic acid can be detected in its position in the sample. In other words, the spatial position (or location) of the nucleic acid in the sample can be determined (or "detected"). This means that the nucleic acid can be located in the cell where it is located or expressed, or in the position of the cell or tissue sample. Therefore, "localization detection" can include determining, measuring, evaluating or analyzing the presence, amount and position of nucleic acids in any way, or not.

[0118] In a specific embodiment, the method can be used for localization detection, especially in situ detection of target nucleic acid sequences. More specifically, the method can be used for localization detection, especially in situ detection of nucleic acids, especially mRNA, in cell samples.

[0119] As used herein, the term "in situ" refers to detecting a target nucleic acid sequence in its natural environment, i.e., in a cell or tissue in which it is normally present. Thus, this may refer to the natural or native location of a target nucleic acid sequence (e.g., RNA). Typically, the term refers to the presence of a nucleic acid in a cell or in a cell or tissue sample, such as its native location in a cell or tissue and / or its location in its normal or native cellular environment.

[0120] In other embodiments, as described above, detection is not localized, or is not original position. In other embodiments, the method can be carried out in solution. In particular, the nucleic acid can be in solution. Therefore, for example, the method can be carried out on a sample comprising isolated nucleic acid.

[0121] The target nucleic acid molecule is present in a sample. The sample can be any sample comprising any amount of nucleic acid from any source or any origin, in which it is desired to detect the target nucleic acid molecule. The sample can be any clinical or nonclinical sample, and can be any biological, clinical or environmental sample in which the target nucleic acid molecule may be present.

[0122] The sample can be any sample containing the target nucleic acid molecule, including natural and synthetic samples, i.e., naturally occurring materials or prepared materials. Naturally occurring samples can be treated or processed before the methods described herein are performed. All biological and clinical samples include, for example, any cell or tissue sample of an organism, or any body fluid or a sample prepared therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples, such as soil and water samples or food samples are also included. The sample can be freshly prepared, or can be pre-treated in any convenient manner, for example, for storage.

[0123] Therefore, representative samples include any material that may contain target nucleic acid molecules, such as food and related products, clinical and environmental samples. The sample can contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Therefore, this biological material can include all types of mammalian and non-mammalian cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, etc., or viruses. Cells can be, for example, human cells, bird cells, reptile cells, etc., without limitation.

[0124] Thus, representative samples include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, stool, cerebrospinal fluid or any other body fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, biopsy, cell culture, cell suspension, conditioned medium or other cell culture component samples, etc. The sample may be pre-treated in any convenient or desired manner in preparation for use in the method, such as by cell lysis or purification, nucleic acid isolation, etc.

[0125] In one embodiment, the sample comprises a microbial cell or virus isolated from a clinical sample or a clinical sample culture. In such a sample, the target nucleic acid molecule can be a nucleotide sequence present in the microbial cell or virus, such as a nucleotide sequence that is characteristic, distinguishing or identifying to the microbial cell or virus at the type, group, class, genus, species or strain level.

[0126] 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 separated. The cell-free DNA may contain circulating tumor DNA.

[0127] For localization in situ detection, sample can be any cell sample that may contain nucleic acid molecules, as long as this sample is suitable for localization in situ detection.The sample can be a sample of a nucleic acid present in a fixed, detectable or visual position in the sample.Therefore, sample will be any sample reflecting normal or native (" in situ ") localization of nucleic acid (such as RNA), i.e. any sample of its normal or native existence.This sample will advantageously be or comprise cell or cell group, such as tissue.For example, sample can be a cell or tissue sample cultivated, harvested or biopsied, wherein nucleic acid can be detected to reveal the qualitative properties of nucleic acid, i.e. its existence, or the existence and / or information of one or more nucleotides in the nucleotide sequence of nucleic acid or nucleic acid, and the location relative to other characteristics of cell.Cell sample can be freshly prepared, or can be pre-treated in any convenient way, such as by fixing or freezing.Therefore, fresh, frozen or fixed cells or tissues, such as FFPE (formalin fixed paraffin embedded) tissues can be used.Sample can comprise any cell type containing nucleic acid, including all types of cells mentioned above.

[0128] The sample may also be treated to fix nucleic acids such as RNA in the cell, for example, to a cell matrix. These procedures are known and described in the art. For example, in the field of in situ hybridization, reagents are known for fixing mRNA to cells. In particular, 5' phosphate groups in RNA can be linked to amines on proteins in the cell matrix via EDC-mediated coupling (EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), thereby helping to maintain the localization of the RNA relative to other cellular components. This technique has been previously described for microRNAs and their detection by in situ hybridization. Alternatively, in a procedure in which a reverse transcription step is performed to generate cDNA from an RNA target, the 5' end of the cDNA primer and / or the cDNA molecule may have cross-linking activity. This can be achieved, for example, by DSP chemicals or NHS acrylates.

[0129] Since the target nucleic acid molecule itself need not be the target analyte of detection, but can be, for example, a reporter molecule used or generated in the process of detecting any desired analyte, the sample need not be a sample naturally containing nucleic acid or nucleic acid source (e.g., cell or virus, or biological or clinical material, etc.). As described above, the sample can be a synthetic or artificial sample. Therefore, it can be a sample that has been tested for the detection of an analyte, wherein a target nucleic acid has been generated or a target nucleic acid molecule has been added thereto. It can be a reaction mixture or a reaction product, such as an immunoassay product for detecting a target analyte, such as immune PCR, immune RCA, or proximity detection (e.g., proximity ligation detection (PLA) or proximity extension detection (PEA), as described above.

[0130] The target analyte can be any analyte that is desired to be detected. As mentioned above, in certain embodiments, the target nucleic acid molecule in the method herein is a target analyte. In other embodiments, when the target nucleic acid molecule is a reporter molecule, the target analyte can be any analyte that is desired to be detected. The analyte can be a nucleic acid, a protein (this term includes peptides and polypeptides), or any other chemical or biological molecule or part, including, for example, carbohydrates, such as carbohydrates that may appear as glycosyl groups on proteins. Therefore, the target analyte can be a modified protein, for example, a protein with a post-translational modification detected in the analyte detection.

[0131] In one embodiment, the target analyte can be a protein or a portion of a protein molecule that is detected on the surface of a cell, vesicle, or other cellular or subcellular compartment. For example, extracellular vesicles or exosomes can be detected and distinguished by the presence of different proteins on their surface.

[0132] As mentioned above, the padlock probe can comprise one or more further sequences, which can be used for introducing a sequence into the connection product, thereby being introduced into RCP (as a complementary copy). This can be, for example, a detection sequence, such as a barcode or an identification motif, or a detection probe or primer binding site. Such a further sequence can be present in a part of the backbone region of the padlock probe, i.e., the region between the target binding regions. In a dumbbell probe, it may be located at the double-stranded region of the probe. Labels such as barcodes or probe / primer binding sites can be designed according to different needs / purposes, for example, introduce universal or common sequences, so that different connected probes are processed together in multiplex settings, for example, to introduce the binding site of universal or common amplification primers. This will enable different connected probes to be amplified together, for example, in the library amplification performed by PCR or RCA.

[0133] In particular, the padlock probe may comprise a detection sequence by which the padlock probe may be detected. The complementary sequence of the detection sequence will be incorporated into the RCP and may be detected by, for example, the binding of the detection probe to it or by sequencing. The detection sequence may be padlock probe specific, thereby being specific to the target sequence or sequence variation that is desired to be detected. Therefore, each padlock probe may have a different detection sequence. The detection sequence may be detected to detect or identify the padlock probe amplified in the RCA, thereby determining the target sequence present. For example, this protocol may be applied to a method for detecting a variation in a target sequence, wherein each padlock probe provides a detection sequence specific to a particular variation. Therefore, the detection sequence may be considered as a marker or recognition sequence. The term "detection sequence" as used herein includes a detection sequence in a padlock probe and a complementary copy that occurs in the RCP.

[0134] Thus, the detection sequence can be used to "tag" or label different padlock probes so that they or their ligations or amplification products can be easily distinguished. In addition, such sequences can be used to label different samples, etc., for example so that they can be combined (i.e., "sample" tags or labels). Thus, in a multiplex setup, different probes (i.e., probes for different target nucleic acid sequences or different variations) can be equipped with different tag sequences (e.g., different labels or detection sequences) and / or they can be equipped with the same tag sequence, for example, for the introduction of a common or universal sequence. These methods can be used in combination with specific detection methods, including the use of detection probes or sequencing methods, such as hybridization sequencing, ligation sequencing or other next-generation sequencing chemistries, for example, multiplex detection of multiple target nucleic acids in a sample.

[0135] The term "hybridization" as used herein refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing or any similar base pairing interaction. When two nucleotide sequences share base pair organizational homology, they are "complementary" to each other. Therefore, a complementary region in a molecule, probe or sequence refers to a portion of the molecule, probe or sequence that is capable of forming a duplex. Hybridization does not require 100% complementarity between sequences, so the complementary region does not require that the sequences are completely complementary, although this is not excluded. Therefore, the complementary region may contain one or more mismatches. Therefore, "complementary" as used herein means "functionally complementary", i.e., a degree of complementarity sufficient to mediate effective hybridization, which includes a degree of complementarity less than 100%. By appropriately adjusting the hybridization conditions, the degree of mismatch allowed can be controlled. A technician in the field of nucleic acid technology can determine duplex stability empirically based on the guidance provided in the art, taking into account a variety of variables, such as the length and base pair composition, ionic strength, and the incidence of mismatched base pairs of each molecule or probe oligonucleotide. Therefore, designing appropriate probes and their binding regions, as well as the conditions for their hybridization with their respective targets, is completely within the scope of the routine skills of those skilled in the art.

[0136] The complementary region, such as the target sequence in the padlock probe binding region, or the complementary region between the detection sequence and the detection oligonucleotide, or the RCA primer and the circularized padlock probe, can be at least 6 nucleotides long to ensure the specificity of the binding, or more specifically, at least 7, 8, 9 or 10 nucleotides long. The upper limit of the region length is not critical, but for example can be up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. Therefore, the length of the complementary region can be in the range between any lower limit and the upper limit mentioned above. In the case of the padlock probe, the length of a single target binding region may be in a lower range, so when the two binding regions are hybridized with the target, the total length is within the upper limit. For example, a single target binding region can be 8-15 nucleotides, such as 10-12 nucleotides, so that the total hybridization length is 16-30 nucleotides long, such as 20-24 nucleotides. Under the constraints of probe conformation, domain spacing, and expectation or preferential hybridization, it may be desirable to minimize the total length of the padlock probe to minimize the size of the ring for RCA, thereby minimizing the length of the complementary region as much as possible.

[0137] RCP may be detected using any convenient protocol.Depending on the target molecule to be detected, the purpose of the method and / or the specific details of the procedure employed in the method, the detection method employed may detect RCP non-specifically or specifically.

[0138] For example, the RCP can be detected directly, for example, the concatemer can be cleaved to generate monomers, which can be detected using gel electrophoresis, or more generally by hybridizing a labeled detection oligonucleotide that hybridizes to a detection sequence in the RCP, as discussed above. However, the detection oligonucleotide does not need to be directly labeled. For example, the detection oligonucleotide can be an unlabeled probe used 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 be bound to the RCP, but they are not directly labeled themselves; instead, they contain a sequence to which a labeled secondary oligonucleotide can bind, thereby forming a "sandwich" between the RCP and the labeled secondary oligonucleotide.

[0139] RCPs can also be detected using non-sequence specific nucleic acid labeling methods, such as DNA binding stains or dyes widely known in the literature, or by incorporating labeled nucleotides into the RCPs. Alternatively, RCPs can be detected indirectly, such as by amplifying the product through PCR and detecting the amplified product.

[0140] RCPs can be detected using any well-established nucleic acid molecule analysis method known in the literature, including liquid chromatography, electrophoresis, mass spectrometry (including CyTOF), microscopy, real-time PCR, fluorescent probes, microarrays, colorimetric analysis (such as ELISA), flow cytometry, mass spectrometry, or by turbidity, magnetic, particle counting, electrical, surface sensing, weight-based detection techniques. In general, these techniques are associated with solution detection.

[0141] However, advantageously, RCP can be detected using labeled detection oligonucleotides, i.e., detection oligonucleotides equipped with a detection moiety. The detection moiety is any detectable moiety, i.e., a moiety that can directly or indirectly generate a detectable signal. Therefore, the detection moiety can be considered as any detectable label, which can directly or indirectly generate a signal. For example, the detection moiety can be spectrally or microscopically detectable, for example, it can be a fluorescent or colorimetric label, a particle (e.g., a magnetic bead) or an enzyme label. Any label used in immunohistochemical techniques can be used.

[0142] In multiplex procedures for detecting different target sequences and / or variant target sequences, different RCP products can be detected and distinguished by in situ sequencing, including, for example, sequencing by synthesis, sequencing by hybridization and 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, combinatorial labeling methods can be used, such as by ratio labeling of fluorescent or other spectrophotometrically detectable oligonucleotides according to techniques well known in the art. Such ratio-labeled detection probes can be used in flow cytometry or microscopy detection techniques (e.g., imaging) to detect a large number of sequences, for example, a combination of at least two fluorophores in different ratios can produce a plurality of fluorescently labeled populations.

[0143] In the method involving the use of detection oligonucleotides, the detection oligonucleotide or any secondary label probe can be labeled with a directly or indirectly detectable label. A directly detectable label is a label that can be directly detected without the need for additional reagents, and an indirectly detectable label is a label that can be detected by using one or more additional reagents, for example, when the label is a member of a signal generation system consisting of two or more components. In many embodiments, the label is a directly detectable label, wherein the relevant directly detectable label includes but is not limited to: fluorescent label, colored label, radioisotope label, chemiluminescent label, etc. In many embodiments, the label is a fluorescent label, wherein the labeling agent used in these embodiments is a fluorescently labeled nucleotide, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP) etc. The fluorescent moiety that can be used for labeling nucleotides to produce a labeled probe nucleic acid (i.e., a detection probe) includes but is not limited to: fluorescein, cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy 630 / 650, etc. Other labels can also be used, such as the above-mentioned labels, which are known in the art.

[0144] Conveniently, for detection in a solution phase method, the detection moiety can be a colored magnetic bead. Colored magnetic beads can be easily visualized. Such magnetic beads, such as colored polystyrene magnetic beads, are widely available.

[0145] Although various detection modes can be used, conveniently, RCP can be detected by visualization, including by microscopy or flow cytometry. In both cases, detection oligonucleotides can be used that are directly or indirectly labeled, for example using fluorescent or colored labels that are easily detected. In this regard, the label can include magnetic beads or other detectable particles. In microscopy-based methods, RCP can be detected by imaging.

[0146] As described above, the present invention also provides a kit for performing the method. The kit may include a padlock probe and a silencing oligonucleotide as described above, optionally including one or more reagents and / or instructions for using the kit. These reagents include dNTPs, polymerases and ligases, and RCA primers for RCA reactions. In addition, the assembly may include a buffer or other reaction assembly for one or more various reactions. Further optional assemblies may include means or reagents for detecting RCP. This may include, for example, detection oligonucleotides and any necessary secondary labeling reagents, including reagents as described above. Further optional assemblies may include solid supports and / or methods for capturing and / or fixing target nucleic acid molecules or reaction assemblies. Instructions may be provided in printed form, on a computer readable medium, or as a website address.

[0147] As described above, the method may be performed using a solid phase, for example, wherein the RCA product is immobilized on a solid phase. This may be due to immobilization of the target molecule, for example in an in situ detection procedure.

[0148] According to principles well known in the art, the fixation of the first RCA product and / or the target molecule on the solid phase can be achieved in a variety of ways. Therefore, various embodiments of solid phase detection are envisioned. In one such embodiment, the sample can be provided on a solid support, such as in an in situ application. Alternatively, the target nucleic acid molecule can be captured by a fixed (or fixable) capture probe, and an RCA product can be generated to attach it to the analyte, such as by the primer of the RCA being the target molecule or attached to the target molecule. Alternatively, the RCA product can be simply fixed on a solid support. For example, the primer of the first RCA can be equipped with a fixable group or part or fixation means, or can be fixed before the first RCA.

[0149] As mentioned above, the target nucleic acid itself may be fixed to a solid phase, for example by non-specific adsorption. In a particular such embodiment, the molecule may be present in cells, optionally fixed and / or permeabilized, and these cells are attached to a solid support, for example a tissue sample containing the target molecule may be fixed on a microscope slide.

[0150] The advantages of the method herein are as described above. These advantages are particularly beneficial in promoting the performance of in-solution detection. By avoiding washing steps or exonucleases to clean up unhybridized probes, a simplified detection scheme can be provided. In particular, the method can be easily performed in a single reaction vessel, such as a single test tube or reaction well. Therefore, by adding reagents to the reaction vessel, the method can be quickly performed within a few minutes. In addition, RCP can be detected in a single container, for example by using a detection oligonucleotide equipped with an easily visualized detection portion (such as a colored magnetic bead). In this way, a direct visual readout can be obtained, for example to detect the presence or absence of a target nucleic acid.

[0151] This approach is well suited for clinical diagnostic testing. This includes enabling home testing of diagnostic tests, or for point-of-care use without the need for a clinical diagnostic laboratory.

[0152] For example, the method can be used to detect the presence of a pathogen in a clinical sample, for example by detecting the presence or absence of pathogen nucleic acid. This may include detecting the presence of a virus, particularly SARS-CoV2, in a sample such as saliva or a nasal or oral swab.

[0153] Furthermore, the technique is rapid and has low instrumentation requirements and allows for multiplexed analysis of sequence variants to increase sensitivity.

[0154] Example

[0155] This method is now described in more detail in the following non-limiting examples.

[0156] Example 1 - Model system

[0157] method

[0158] 1) Prepare synthetic templates representing the target SARS-CoV-2 genome containing 9 different target sequences. Add the template to the test tube in a 20 μl volume and a concentration of 10 pM. Prepare a control test tube that contains no template but instead contains the same amount of "wrong template" (i.e., a synthetic non-target template molecule, representing a "negative sample test tube").

[0159] 2) Prepare a ligation mixture by mixing 5 nM of each padlock probe (9 different padlock probes targeting different sequences of the target genome), 1-5 nM of each silencing oligonucleotide (1 silencing oligonucleotide for each padlock probe), and ligase. Add 5 μl of the ligation mixture to the template. Incubate the tube at room temperature for 5 minutes.

[0160] 1. Alternative 1 Mix padlock probe and ligase and add to template. Add silencing oligonucleotide and incubate at room temperature for 5 minutes.

[0161] 2. Alternative 2 Add the padlock probe to the template, then add the ligase, followed by the silencing oligonucleotide. Incubate at room temperature for 5 minutes.

[0162] 3) Perform RCA reaction. Prepare the components required for RCA reaction by adding detection beads and Phi29 polymerase to the RCA mixture containing BSA, Phi29 buffer and dNTPs. Add 15 μl of RCA mixture to the ligation reaction and incubate the tube at 37°C for 25 minutes. Detection beads are functionalized colored magnetic beads carrying detection oligonucleotides.

[0163] 4) Read the results by visual inspection of the test tube.

[0164] result

[0165] like Figure 3 As shown, the sample tube containing the synthesized target template reported a positive result (above). The positive result can be seen by observing the obvious colored RCA reaction product at the bottom of the tube (caused by the aggregation of colored detection beads in the RCP at the bottom of the tube). The test tube that did not contain the synthesized target template (i.e., the test tube containing the wrong template) reported a negative result; no aggregation of colored magnetic beads in the RCA reaction product was observed, and the colored detection beads were evenly distributed in the reaction solution in the test tube.

[0166] To demonstrate the reproducibility of the method, the experiment was repeated to compare the synthetic templates representing the correct target nucleic acid molecule (4 replicates) with the control template lacking the padlock probe target sequence, which represents the incorrect target molecule (4 replicates). Figure 4 A. Positive results are seen with the correct target molecule, while negative results are seen with the wrong target molecule. This shows that the specificity of the detection test can be controlled by using silencing oligonucleotides.

[0167] The titration results of different amounts of silencing oligonucleotides are shown in Figure 4 As shown in B. This shows that at 5, 4.5, 4 and 3.5 nM silencing oligonucleotide concentrations, a clear positive reaction can be clearly observed. The RCA reaction product is still visible at 3 nM, but it is not obvious at 2.5 nM. At 2 and 1.5 nM silencing oligonucleotide concentrations, no obvious RCA reaction product is seen. This shows that the presence of a sufficient amount of silencing oligonucleotide allows the RCA reaction product to be visualized. This shows that a person skilled in the art can easily determine the appropriate ratio between the padlock probe and the silencing oligonucleotide and the template.

[0168] Example 2 - Clinical Sample Testing

[0169] method

[0170] 1) Obtain clinical samples that are confirmed COVID-19 positive or negative by PCR. Dilute the samples into the appropriate carrier buffer, which is provided and prepared in 20 μl aliquots, and add to the test tubes. Test tubes 1-20 contain samples that are COVID-19 PCR positive, and test tubes 21-40 contain samples that are COVID-19 PCR negative.

[0171] 2) Prepare a ligation mixture by mixing 5 nM of each padlock probe (9 different padlock probes targeting different sequences of the target genome), 1-5 nM of each silencing oligonucleotide (1 silencing oligonucleotide for each padlock probe), and ligase. Add 5 μl of the ligation mixture to the template. Incubate the tube at room temperature for 5 minutes. For comparison, no silencing oligonucleotide was added to the ligation mixture.

[0172] 1. Alternative 1 Mix padlock probe and ligase and add to template. Add silencing oligonucleotide and incubate at room temperature for 5 minutes.

[0173] 2. Alternative 2 Add the padlock probe to the template, then add the ligase, followed by the silencing oligonucleotide. Incubate at room temperature for 5 minutes.

[0174] 3) Perform RCA reaction. Prepare the components required for RCA reaction by adding detection beads and Phi29 polymerase to the RCA mixture containing BSA, Phi29 buffer and dNTPs. Add 15 μl of RCA mixture to the ligation reaction and incubate the tube at 37°C for 25 minutes. Detection beads are functionalized colored magnetic beads carrying detection oligonucleotides.

[0175] 4) Read the results by visual inspection of the test tube.

[0176] result

[0177] Figure 5 Results of experiments without (A) and with (B) the silencing oligonucleotide in the ligation mixture are shown. As can be seen, in the absence of the silencing oligonucleotide, there is no difference between the COVID-19 positive tubes (tubes 1-20) and the COVID-19 negative tubes (tubes 21-40). In contrast, in the presence of the silencing oligonucleotide, positive results (presence of RCA reaction products) can be detected in the COVID positive tubes. Regarding tubes 17-20 in (B), although the results are not as clear as in the other tubes, aggregation of colored magnetic beads can be seen, indicating the presence of RCA reaction products, in contrast to tubes 21-32.

[0178] Example 3 - Demonstration of sensitivity improvement using a model system

[0179] Experiments were performed using the model system described in Example 1, either in the presence or absence of silencing oligonucleotides.

[0180] The results are as follows Figure 6Figures 2A and 2B show the results of a model system that uses a triple-binding silencing oligonucleotide to obtain improved sensitivity. (A) shows the titration results of the template oligonucleotide in the model system in the absence of the silencing oligonucleotide. (A) shows the template oligonucleotide at concentrations of 5nM, 500pM, 50pM, 5pM, 500fM, 50fM, and 5fM, as well as a negative control (Neg C), and shows the binding of different detection oligonucleotides to the rolling circle product (RCP) or free padlock probe. In this experiment, the free padlock probe was not inhibited from interacting with the RCP by the silencing oligonucleotide. The detection limit of this experiment was between 5pM and 50pM. (B) shows the titration results of the triple-binding silencing oligonucleotide and the template oligonucleotide in the model system. (B) shows the template oligonucleotide at concentrations of 5nM, 500pM, 50pM, 5pM, 500fM, 50fM, and 5fM, as well as a negative control (Neg C), and the silencing oligonucleotide at a 1:1 ratio with the padlock probe. The results showed that the silencing oligonucleotide allowed the RCP to grow without interference from the free padlock probe that bound to the growing padlock probe with one arm. If the padlock probe was allowed to bind with one arm, the RCP would become double-stranded. Only perfect binding of the padlock probe to the growing RCP was possible (because the padlock probe prefers to bind to the perfect target (18+18nt(36nt)) rather than the triply bound silencing oligonucleotide; however, if the free padlock probe bound to the RCP with one arm, the silencing oligonucleotide would bind to the other arm and the phi29 polymerase would not be able to make the product double-stranded), which allowed the generation of the second generation RCP. The silencing oligonucleotide allowed the padlock probe to bind perfectly to the template, thereby increasing the sensitivity of the detection to a detection limit as low as 5fM to 50fM.

[0181] Example 4 - Display of a second round of RCA using triple binding silencing oligonucleotides

[0182] Two different silencing oligonucleotide designs were compared using the model system in Example 1, the first being a silencing oligonucleotide containing only the first and second silencing regions (target binding sites / arms of the targeting padlock probe) separated by a 20 nt spacer sequence; the second being a triple binding silencing oligonucleotide containing a third silencing region.

[0183] These silencing oligonucleotides were tested at different concentrations of template molecules (from 5 nM to 5 fM).

[0184] The results are as follows Figure 7 As shown in parts (A) and (B) of FIG. 1 , it can be seen that in the presence of a triple binding oligonucleotide, as shown in (B), a greater amount of RCA products can be seen, including a lower concentration of template molecules, compared to the double binding silencing oligonucleotide in (A).

[0185] The results show that the triple binding silencing oligonucleotide with three silencing regions allows a second round of RCA to be performed (on top of the first round). Due to the more rigid conformation of the padlock probe / silencing oligonucleotide homologue, which only occurs when the silencing oligonucleotide binds to the padlock probe in three positions, the padlock probe will prefer to bind to the template rather than the silencing oligonucleotide when the correct template sequence is present. The silencing oligonucleotide (A) that does not bind to the padlock probe backbone does not form this more rigid conformation when binding to the padlock probe and therefore does not have this preference for the template sequence. Therefore, (A) does not allow this lower sensitivity - it does not allow a second round of RCA to be performed. The triple binding silencing oligonucleotide allows the padlock probe to perfectly bind to the template - firstly the template of the target molecule (such as the viral genome), and secondly the growing RCP containing the target sequence of the initial target molecule - which improves the sensitivity of the detection.

[0186] Example 5 - Study on the design of different triple binding silencing oligonucleotides

[0187] Two silencing oligonucleotide designs were tested in the model system of Example 1, such as Figure 8 As shown, they are called Danish configuration (BD) and Omega configuration (BO), respectively.

[0188] Different lengths of the first, second and third silencing regions were tested, ranging from 6-18 for the first and second silencing regions ("arms") and 2-30 for the third silencing region ("backbone", Bp).

[0189] The results are shown in Table 1 below:

[0191] Table 1

[0192]

[0193] The results can be summarized as ΔG(pad-temp)<ΔG(pad-block)<ΔG(pad);

[0194] -56<ΔG(pad-block)<-17.

[0195] Silencing oligonucleotides with a first-second-third silencing region of 18-20-18 or 10-20-10, where the 10 nt first and second silencing regions leave the target binding site of the padlock probe closest to the ligation site vacant, are very efficient.

[0196] Other variants are less efficient, but they are still effective enough to work effectively.

Claims

1. A method for detecting a target nucleic acid molecule in a sample, the method comprising: (i) contacting the sample with a padlock probe comprising target binding regions at its 5' and 3' ends that are complementary to a probe binding site 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 region at the end of the padlock probe, wherein the two silencing regions are separated from each other by at least 5 nucleotides, and wherein the contacting occurs before, during or after contacting the sample with the padlock probe; (iii) hybridizing and juxtaposing the target-specific binding region of the padlock probe and the target nucleic acid molecule so as to directly or indirectly link 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 is contacted with 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 an RCA template 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 performed in a solution.

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 molecule for a target analyte.

4. The method of any one of claims 1 to 3, wherein the padlock probe is pre-hybridized with the silencing oligonucleotide, and the target nucleic acid molecule displaces the silencing oligonucleotide from the padlock probe hybridized with the target nucleic acid molecule.

5. The method according to any one of claims 1 to 3, wherein the padlock probe and the silencing oligonucleotide are contacted with the sample containing the target nucleic acid molecule simultaneously or in the form of a mixture.

6. The method of any one of claims 1 to 3, wherein after hybridizing the padlock probe to the target nucleic acid molecule, or after ligation step (iv), the silencing oligonucleotide is contacted with the sample to allow it to hybridize with any unbound or unligated padlock probe in the sample.

7. The method of any one of claims 1 to 6, wherein the silencing region is located at the 5' and 3' ends of the silencing oligonucleotide.

8. The method of any one of claims 1 to 7, wherein the silencing oligonucleotide comprises a third silencing region, wherein the third silencing region is located between the first and second silencing regions and is complementary to the silencing oligonucleotide binding site in the padlock probe, wherein the silencing oligonucleotide binding site is located inside the padlock probe, between its 5' end and 3' end.

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

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

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

13. The method according to any one of claims 9 to 12, wherein the detection sequence partially or completely overlaps with the silencing oligonucleotide binding site, and the silencing oligonucleotide binding site is complementary to the third silencing region of the silencing oligonucleotide.

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

15. The process of any one of claims 1 to 14, wherein the process is performed in a single reaction vessel.

16. 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 a probe binding site in the target nucleic acid molecule; and (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.

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

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

19. A silencing oligonucleotide for blocking the target binding region of a padlock probe, the silencing oligonucleotide comprising a first and a second silencing region 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, and wherein the silencing oligonucleotide further comprises a third silencing region, wherein the third silencing region is located between the first and second silencing regions and is complementary to the silencing oligonucleotide binding site in the padlock probe located between the target binding regions of the padlock probe.

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