WO2025006625A1

WO2025006625A1 - Interlocking nucleic acids for target hybridization

Info

Publication number: WO2025006625A1
Application number: PCT/US2024/035646
Authority: WO
Inventors: Esther Musgrave-Brown; Carlo RANDISE-HINCHLIFF; Niall Anthony Gormley
Original assignee: Illumina Inc
Current assignee: Illumina Inc
Priority date: 2023-06-26
Filing date: 2024-06-26
Publication date: 2025-01-02
Anticipated expiration: 2025-12-26

Abstract

Interlocking nucleic acids for target hybridization are described. The interlocking nucleic acids include individual nucleic acids having respective capture regions that hybridize to the target nucleic acid and interlocking regions that bind to each other. In an embodiment, the interlocking nucleic acids are part of a probe set that specifically binds to a target nucleic acid to permit capture of the target nucleic acid. In an embodiment, the interlocking nucleic acids may be part of a primer used for amplification of the target nucleic acid.

Description

INTERLOCKING NUCLEIC ACIDS FOR TARGET HYBRIDIZATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. Provisional Application No. 63/510,212, entitled “INTERLOCKING NUCLEIC ACIDS FOR TARGET HYBRIDIZATION,” and filed on lune 26, 2023.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on June 25, 2024, is named “ILUM0103PCT.xmr’ and is 19,229 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

BACKGROUND

[0003] The disclosed technology relates generally to nucleic acids such as probes and/or primers that specifically bind to target sequences. In particular, the technology disclosed relates to nucleic acid assemblies formed from interlocking or partially self-complementary nucleic acids having regions that hybridize to a target nucleic acid as well as regions that hybridize to an adjacent nucleic acid of the nucleic acid assembly.

[0004] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology. [0005] Sequencing methodology of next-generation sequencing (NGS) platforms typically makes use of nucleic acid fragment libraries. In targeted sequencing techniques, a subset of fragments containing genes or regions of interest of the genome are isolated from the nucleic acid library and sequenced. Targeted approaches using NGS allow researchers to focus time, expenses, and data analysis on specific areas of interest. Such targeted analysis can include the exome (the protein-coding portion of the genome), specific genes of interest (custom content), targets within genes, or mitochondrial DNA. Targeted approaches contrast with whole genome sequencing approaches that are more comprehensive, but that also involve sequencing regions of the genome that may not be of interest to all users.

[0006] In one example of a targeted sequencing technique, target enrichment or hybridization pullout methods use a panel or set of probes that hybridize to target sequences in the nucleic acid library. Hybridization of the probes to the target sequences allows these sequences to be separated from the rest of the fragments in the library to enrich the targeted sequencing using the captured sequences.

[0007] NGS platforms and other nucleic acid analysis techniques may also include one or more amplification reaction that use primers that are specific for a target sequence. Such primers are typically shorter than the probes used in hybridization pullouts. However, desired specificity of the primers for target sequences is achieved by the user of primer pairs that are specific for sequences at opposing ends of the target.

BRIEF DESCRIPTION

[0008] In one embodiment, the present disclosure provides a probe set for a target nucleic acid sequence. The probe set includes a first single-stranded oligonucleotide probe comprising a first probe capture region and a first probe 5’ interlocking region; a second single-stranded oligonucleotide probe comprising a second probe 3’ interlocking region complementary to the first probe 5’ interlocking region, a second probe 5’ interlocking region, and a second probe capture region; and a third single-stranded oligonucleotide probe comprising a third probe capture region and a third probe 3’ interlocking region complementary to the second probe 5’ interlocking region.

[0009] In one embodiment, the present disclosure provides a primer composition. The primer composition includes a first single-stranded oligonucleotide comprising a first hybridization region complementary to first nucleotides of a target nucleic acid; and a first interlocking region 3’ of the first hybridization region. The primer composition also includes a second single-stranded oligonucleotide comprising a second hybridization region complementary to second nucleotides of the target nucleic acid, wherein the second nucleotides are 5’ of the first nucleotides on the target nucleic acid; and a second interlocking region 5’ of the second hybridization region and complementary to the first interlocking region.

[0010] In one embodiment, the present disclosure provides a method that includes the steps of fragmenting nucleic acids of a sample to generate nucleic acid fragments comprising target sequences and contacting the nucleic acid fragments with a plurality of probes, wherein the plurality of probes comprises individual probe sets specific for respective target sequences. An individual probe set includes a first oligonucleotide probe comprising comprising a first probe capture region specific for a first portion of a target sequence of the nucleic acid fragments and a first probe 5’ interlocking region ; a second oligonucleotide probe comprising a second probe 3’ interlocking region complementary to the first probe 5’ interlocking region, a second probe 5’ interlocking region, and a second probe capture region specific for a second portion of a target sequence; and a third oligonucleotide probe comprising a third probe capture region specific for a third portion of a target sequence and a third probe 3’ interlocking region complementary to a second probe 5’ region, and wherein the contacting is under conditions allowing for hybridization of the individual probe sets to the nucleic acid fragments and interlocking of the individual probe sets to form interlocked probe-fragment complexes, and wherein the contacting is under conditions allowing for hybridization of the individual probe sets to the nucleic acid fragments and interlocking of the individual probe sets to form interlocked probe-fragment complexes. The method also includes separating the interlocked probe-target complexes from unhybridized nucleic acid fragments of the nucleic acid fragments to generate separated nucleic acid fragments.

[0011] In one embodiment, the present disclosure provides a method that includes the steps of contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; and detecting the analytes by detecting aptamers of the analyte-aptamer complexes. Detecting the analytes includes contacting the individual aptamer with an interlocking probe set. The interlocking probe set includes a first single-stranded oligonucleotide probe comprising a first complementary region that hybridizes to a first region of the individual aptamer and a first interlocking region; and a second single-stranded oligonucleotide probe comprising a second complementary region that hybridizes to a second region of the individual aptamer, a nonhybridizing region comprising an identification sequence uniquely identifying for the individual aptamer, and a second interlocking region, wherein the first interlocking region and the second interlocking region hybridize to one another. The detecting also includes capturing a first probe of probe set via binding of the affinity tag to an affinity tag binder to capture the individual aptamer and the second probe hybridized to the second region of the individual aptamer; and detecting the identification sequence of the captured second probe.

[0012] In one embodiment, the present disclosure provides a method that includes the steps of contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; and detecting the analytes by detecting aptamers of the analyte-aptamer complexes. Detecting an individual aptamer of the plurality of aptamers comprises contacting the individual aptamer with a reporter probe and an interlocking probe set to cause the aptamer, the interlocking probe set, and the reporter probe form a complex and detecting an identification sequence of the reporter probe of the complex. The interlocking probe set comprises: a first single-stranded oligonucleotide probe comprising a first aptamer complementary region that hybridizes to a first region of the individual aptamer, a first reporter probe complementary region that hybridizes to a first region of the reporter probe, and a first interlocking region; and a second single-stranded oligonucleotide probe comprising a second complementary region that hybridizes to a second region of the individual aptamer, a second reporter probe complementary region that hybridizes to a second region of the reporter probe, and a second interlocking region, wherein the reporter probe comprises an identification sequence uniquely identifying for the individual aptamer, and wherein the first interlocking region and the second interlocking region hybridize to one another in the complex

[0013] The preceding description is presented to enable the making and use of the technology disclosed. Various modifications to the disclosed implementations will be apparent, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the technology disclosed. Thus, the technology disclosed is not intended to be limited to the implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. The scope of the technology disclosed is defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0015] FIG. l is a schematic illustration of an example interlocked probe set bound to a target nucleic acid compared to a conventional probe, in accordance with aspects of the present disclosure;

[0016] FIG. 2 is a schematic illustration of binding and assembly of an interlocked probe set on a target binding region of a target nucleic acid, in accordance with aspects of the present disclosure; [0017] FIG. 3 is a schematic illustration of an alternate binding order of an interlocked probe set during assembly on target binding region of a target nucleic acid, in accordance with aspects of the present disclosure;

[0018] FIG. 4 is a schematic illustration of an individual nucleic acid of the interlocked probe set of FIG. 1 bound to an off-target nucleic acid;

[0019] FIG. 5 is a schematic illustration of an example interlocked probe set bound to a target nucleic acid, in accordance with aspects of the present disclosure;

[0020] FIG. 6 is a schematic illustration of a partially assembled probe nonspecifically bound to an off-target nucleic acid sequence;

[0021] FIG. 7 shows an example target enrichment workflow using interlocked probe sets for enrichment targets, in accordance with aspects of the present disclosure;

[0022] FIG. 8 shows an example first cycle amplification using a locked forward primer, in accordance with aspects of the present disclosure;

[0023] FIG. 9 shows an example first cycle amplification using a locked reverse primer, in accordance with aspects of the present disclosure;

[0024] FIG. 10 shows an example second cycle amplification using a locked forward primer, in accordance with aspects of the present disclosure;

[0025] FIG. 11 shows an example second cycle amplification using a locked reverse primer, in accordance with aspects of the present disclosure;

[0026] FIG. 12 shows subsequent cycle amplifications using modified extended primers after initial cycles using locked forward and reverse primers, in accordance with aspects of the present disclosure; [0027] FIG. 13 shows an example workflow for a tri-molecular assay for aptamer detection used in conjunction an interlocking probe set, in accordance with aspects of the present disclosure;

[0028] FIG. 14 shows a relationship with melting temperature and probe performance;

[0029] FIG. 15 shows an example arrangement of a probe set for use in the assay of FIG. 13, in accordance with aspects of the present disclosure;

[0030] FIG. 16 shows an example arrangement of a probe set for use in the assay of FIG. 13 to assess probe performance, in accordance with aspects of the present disclosure;

[0031] FIG. 17 shows an example interlocking probe set that binds an aptamer and a corresponding reporter probe;

[0032] FIG. 18 is a schematic illustration of an example interlocked probe set include a surface-linked probe that assembles when bound to a target nucleic acid, in accordance with aspects of the present disclosure;

[0033] FIG. 19 shows an example heteroallele readout using the probe set of FIG. 18;

[0034] FIG. 20 shows an example homoallele readout using the probe set of FIG. 18;

[0035] FIG. 21 shows an example homoallele readout using the probe set of FIG. 18;

[0036] FIG. 22 shows an example probe hybridization arrangement to detect polymorphic allele variants; and

[0037] FIG. 23 shows an example interlocked probe hybridization arrangement to detect polymorphic allele variants.

DETAILED DESCRIPTION [0038] The following discussion is presented to enable any person skilled in the art to make and use the technology disclosed, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed implementations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the technology disclosed. Thus, the technology disclosed is not intended to be limited to the implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0039] The use of nucleic acids with specificity for a target sequence permits target enrichment and/or amplification. Relatively longer nucleic acids can provide greater target specificity relative to shorter nucleic acids and with reduced incidence of off-target binding. The binding strength of a nucleic acid for a target increases with an increasing number of complementary nucleotides. However, the total target annealing time is a function of nucleotide length of the annealing nucleic acid. Thus, longer nucleic acids may have much longer target binding times (e.g., 1-2 hours) relative to shorter sequences that specifically bind within minutes, or even seconds, to the target. In addition, longer nucleic acids used for specific binding reactions are relatively more expensive to synthesize. Accordingly, providing reaction workflows for target enrichment/amplification with desired target specificity but without burdensome reaction times or costly components is challenging. Provided herein are nucleic acids that have a target binding specificity that is characteristic of a specificity of relatively longer nucleic acids but with the faster binding kinetics associated with shorter nucleic acids.

[0040] In an embodiment, the disclosed techniques include interlocking hybridization probe sets that, for example, may be used for target enrichment NGS. An individual interlocking probe set includes a plurality of nucleic acids that are, respectively, complementary to different regions of a target sequence. The nucleic acids are also at least in part complementary to one another, such that an individual nucleic acid of the probe set partially binds to the target sequence and partially binds to at least one other nucleic acid of the probe set. In this manner, the probe set is interlocking via its self-complementary regions. The target binding region of each individual nucleic acid of the probe set is relatively shorter (e.g., 10-40 nucleotides in an embodiment) than the binding region of a conventional contiguous or single nucleic acid hybridization probe, which may be 80-300 nucleotides in length.

[0041] The target binding for the interlocking probe set includes multiple shorter binding interactions to correspondingly shorter respective target regions, which means that the interlocking probe set has binding kinetics that more closely resemble the kinetics of shorter nucleotides. Once interlocked, the total length of the target sequence bound by the combination of individual nucleic acids of the probe set is comparable to a total length annealed by conventional probes. The melting temperature of the interlocked probe set is boosted through the interlocking and multiple separate target binding regions, and the interlocked probe set is stably bound at boosted hybridization temperatures at which nonspecific binding is disrupted. Thus, the interlocking probe sets can achieve target specificity at or close to that or a conventional probe with improved binding kinetics characteristics of shorter nucleic acids. Provided herein are probe set compositions, nucleic acids, reagents, kits, probe panels, and methods of manufacturing and using interlocking probe sets. The disclosed interlocking probe sets may be part of an isolated reaction or may be used as part of a larger workflow, such as a sequencing workflow.

[0042] In another embodiment, the disclosed techniques include interlocking primers that may improve primer binding specificity without extending or significantly extending primer annealing time. An individual interlocking primer set includes a plurality of nucleic acids that are, respectively, complementary to different regions of a target sequence. The nucleic acids are also at least in part complementary to one another, such that an individual nucleic acid of the primer set partially binds to the target sequence and partially binds to at least one other nucleic acid of the primer set. The primer set may be part of a primer pair in which one or both primers of the primer pair is part of an interlocking primer set. Provided herein are primer set compositions, primer pairs, nucleic acids, amplification reagents, amplification kits including amplification reagents (e.g., polymerases) and primer compositions, and methods of manufacturing and using interlocking primer sets. The disclosed interlocking primer sets may be part of an isolated amplification reaction or may be used as part of a larger workflow, such as a sequencing workflow. Further, amplification using interlocking primers may include initial cycles using the interlocking primers and subsequent cycles using conventional or noninterlocking primers.

[0043] As provided herein, an interlocking probe set and/or primer set may refer to two or more nucleic acids, whereby each individual nucleic acid of the set has a region of complementarity to a respective portion of the target nucleic acid, e.g., a single-stranded target nucleic acid, and has a region of complementary to at least one other nucleic acid of the set. The interlocking probe and/or primer set may be in an unassembled state, e.g., when stored apart from any target nucleic acid, may be in an assembled state bound to the target nucleic acid, or may be in a transition state during an annealing or denaturing process in which only some of the nucleic acids are bound to the target and/or one another. The nucleic acids of the interlocking prober and/or primer set may be single-stranded when in a fully unassembled state and not bound to the target nucleic acid or to one another. In an embodiment, the nucleic acids of the interlocking prober and/or primer set are in an all or mostly double-stranded arrangement (when bound to the target nucleic acid and one another) when fully assembled. However, in certain embodiments, portions of one or more of the unassembled nucleic acids may be double-stranded. The individual nucleic acids may be coupled to affinity binders or stability linkers that promote an interlocked configuration in an embodiment. The individual nucleic acids may include index sequences or barcodes that are part of the interlocking region or that are on 5’ or 3’ ends of the nucleic acids.

[0044] FIG. 1 is a schematic illustration of an example interlocking probe set 10 according to the disclosed embodiments. The probe set 10 is shown in an interlocked or assembled state and annealed or hybridized to a target nucleic acid 11. The probe set 10, as illustrated, includes three separate nucleic acids 16, 18, 20 that respectively bind to different part or regions of a target binding sequence 24 of the target nucleic acid 11. While the illustrated interlocking probe set 10 includes three nucleic acids, it should be understood that the probe set 10 as provided herein may include two, three, four, five, six, seven, or more individual nucleic acids. Where the probe set includes three, four, five, six, seven, or more nucleic acids, it should be understood that the 5’ terminal and 3’ terminal nucleic acids may include a single interlocking region while interior nucleic acids include both 5’ and 3’ interlocking regions that flank a capture region. Thus, the terminal nucleic acids may differ in both arrangement and number of interlocking regions relative to the interior nucleic acids. Further, it should be understood that the nucleic acids of the interlocking probe set 10 may be in a single-stranded state when not assembled in place on (i.e., annealed to) the target nucleic acid 11 and hybridized to each other. However, the interlocking probe set 10 forms an at least partially double- stranded structure when assembled on the target nucleic acid 11.

[0045] Each of the nucleic acids 16, 18, 20 includes a respective probe capture region 30, 32, 34 that is complementary to only a portion of the target binding sequence 24 and also includes at least one interlocking region 40, 42, 44, 46 that is not complementary to the target binding sequence 24 and that is complementary to another of the interlocking regions 40, 42, 44, 46. In one embodiment, an individual interlocking region of the probe set 10 is complementary to another interlocking region of the probe set 10 based on adjacently-bound capture regions. That is, nucleic acids bound adjacently to one another on the target sequence 24 also include complementary interlocking regions.

[0046] In the illustrated example, a 5’ terminal nucleic acid 20 of the interlocking probe set 10 includes a probe capture region 34 extending from a 5’ end of the nucleic acid and complementary to a 3 ’-most portion of the target binding sequence 24. The 5’ terminal nucleic acid 20 also includes a 3’ interlocking region 46 that is complementary to the 5’ interlocking region 44 of the nucleic acid 18 bound to a neighboring portion of the target binding sequence 24. The interior nucleic acid 18 includes a single probe capture region 32 that is flanked by interlocking regions 42, 44. The 3’ terminal nucleic acid 16 of the interlocking probe set includes an interlocking region 40 at a 5’ end and a probe capture region 30 at a 3’ end that is complementary to a 5’ portion of the target binding sequence 24. In the illustrated example, the nucleic acids of the interlocking probe set 10 assembled on the target nucleic acid 11 to include a 5’-most portion and a 3’-most portion of the assembled probe set 10 that is annealed in place on the target binding sequence 24.

[0047] The interlocking probe set 10 also includes an affinity binder 50 that can be used to separate bound target nucleic acids 11 from unbound fragments. In an embodiment, the affinity binder 50 is coupled to the interior nucleic acid 18, which may be relatively more strongly annealed on the target nucleic acid 11 based on cooperative binding with the end nucleic acids 16, 20 and at temperatures that permit assembly of the probe set 10 on the target nucleic acid 11. These temperatures may be selected so that the probe set 10 remains in place while any nonspecifically bound nucleic acids of other probe sets are separated from off-target binding. Such assembly temperatures are a function of a total length of the probe capture region 30, 32, 34 bound to the target binding sequence 24, a nucleotide composition of the probe set, and the bound interlocking regions 40, 42, 44, 46. Because the disclosed embodiments may include a plurality of different probe sets 10 specific for different target nucleic acids 11, a suitable temperature or temperature range that works for the pool of probe sets 10 may be selected, e.g., from a lower bound of estimated available temperatures that permit binding of the interlocked probe sets 10 but at which individual nucleic acids of the probe sets 10 separate from off-target binding sites.

[0048] The respective lengths of the target-specific capture regions and the interlocking regions of the nucleic acid can be selected to achieve a desired melting temperature (Tm). In an embodiment, the target-specific capture regions of an individual probe set 10 have similar Tms relative to one another of melting or separating from the target sequence 24. Further, in embodiments in which a panel of multiple different probe sets 10 are used (see FIG. 7), all of the different probe sets 10 may be designed such that the all or most of the capture regions of the nucleic acids of each different probe set 10 fall within a preset estimated Tm. In an embodiment, the estimated Tm range for each individual capture region of each nucleic acid is between 45-65°C. In an embodiment, the various interlocking regions of an individual probe set 10 have similar Tms relative to one another of melting or separating from each other. Further, in embodiments in which a panel of multiple different probe sets 10 are used (see FIG. 7), all of the different probe sets 10 may be designed such that all or most of the interlocking regions of each different probe set 10 fall within a preset estimated Tm. In one embodiment, the individual capture region Tms of an individual probe set 10 are higher than the individual interlocking region Tms across the probe set 10. If the interlocking region Tms were about the same as or higher than the capture region Tms, the individual nucleic acids of a probe set 10 would anneal to each other via their locking sequences before they anneal to the target, and this would make them behave similarly to a long probe (with slow hybridization kinetics).

[0049] In an embodiment, the Tm for a particular capture region sequence, an interlocking region sequence, an individual nucleic acid of the probe set 10, or a fully assembled probe set may be estimated based on the following assumptions nearest neighbors formula:

Where:

T_m = melting temperature in °C

A/7 = enthalpy change in kcal mol’¹ (accounts for the energy change during annealing / melting)

A = constant of -0.0108 kcal K ¹ • mol ¹ (accounts for helix initiation during annealing / melting)

AA = entropy change in kcal K ¹ • mol ¹ (accounts for energy unable to do work, i.e. disorder)

R = gas constant of 0.00199 kcal K'¹ • mol'¹ (constant that scales energy to temperature)

C = oligonucleotide concentration in M or mol L'¹ (Table 1 is shown at 0.0000005, i.e. 0.5 pM)

-273.15 = conversion factor to change the expected temperature in Kelvins to °C

[AG ] = sodium ion concentration in M or mol L ¹ (Table 1 is shown at 0.05, i.e. 50 mM) Molecule DNA RNA

Interaction AH A A AH A5

AA/TT* -9.1** -0.0240 -6.6 -0.0184 AT/TA -8.6 -0.0239 -5.7 -0.0155

TA/ AT -6.0 -0.0169 -8.1 -0.0226

CA/GT -5.8 -0.0129 -10.5 -0.0278

GT/CA -6.5 -0.0173 -10.2 -0.0262

CT/GA -7.8 -0.0208 -7.6 -0.0192

GA/CT -5.6 -0.0135 -13.3 -0.0355

CG/GC -11.9 -0.0278 -8.0 -0.0194

GC/CG -11.1 -0.0267 -14.2 -0.0349

GG/CC -11.0 -0.0266 -12.2 -0.0297

Table 1 : *The left sequence is 5' to 3', while the right sequence is 3' to 5', e g. for AA/TT, AA is 5' to 3' and TT is 3' to 5'. When selecting values, always choose in the 5' to 3' direction, regardless of whether it is the left or right sequence in the correct orientation. **Negative values reflect that annealing is enthalpically and entropically favorable. Positive values would reflect the reverse reaction, melting, and would lead to an identical Tm calculation.

[0050] In another example, the Tm may be estimated as follows:

• For sequences less than 14 nucleotides the formula is:

Tm= (wA+xT) * 2 + (yG+zC) * 4 where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

• For sequences longer than 13 nucleotides, the equation used is:

Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

Equations above assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Nay and pH 7.0. In an embodiment, the sequence length is based on a total length of the nucleic acid (e.g., nucleic acids 16, 18, 20).

[0051] In an embodiment, suitable temperatures to reduce nonspecific binding may include temperatures at which the end nucleic acids 16, 20 may start to separate from the target nucleic acid 11. However, even in cases where the end nucleic acids 16, 20 are partially separated from the target nucleic acid 11, the interior nucleic acid 18, after assembly of the probe set 10, may be more stably bound and therefore may remain in place on the target nucleic acid 11 over a temperature transition range that would cause the end nucleic acids 16, 20 to become at least partially single-stranded. In this manner, the affinity binder 50 remains in place on the target nucleic acid 11 at relatively higher temperatures relative to those associated with removal of the end nucleic acids 16, 20 to permit separation and enrichment of the target nucleic acid 11 via the affinity binder 50. This potential upshifting of the available temperatures at which the affinity binder 50 of the probe set 10 remains in place on the target nucleic acid 11 permits an associated broader range of temperatures to be used to separate nonspecific binders from target nucleic acids 11, thus increasing the overall specificity of the reaction while also permitting relatively faster times to complete hybridization. Further, certain probe set arrangements may include end nucleic acids (e.g., nucleic acids 16, 20) with longer capture regions 30, 34 or with capture regions 30, 34 that include modified nucleic acids (e g., locked nucleic acids) to enhance stability and hybridization to the target sequence 24.

[0052] The illustrated discrete probe capture regions 30, 32, 34 are in contrast to the contiguous capture region of a conventional probe 53, which is shown as having specificity for about 80 nucleotides of the target binding sequence 24. The annealing time of the conventional probe 53 is a function of the 80 nucleotide length. However, the interlocking probe set 10 binds according to the annealing time of each individual nucleic acid. By way of example, the individual nucleic acids 16, 18, 20 are shown as having probe capture regions about 30 nucleotides in length, which is significantly shorter than the capture region of the conventional probe 53. It should be understood that the illustrated capture region length is by way of example, and each individual capture region 30, 32, 34 may be between 10-40 nucleotides in one embodiment. Further, the each individual capture region 30, 32, 34 of an interlocking probe set 10 may have about the same length across the set 10 or may have variable lengths relative to one another.

[0053] Because the binding kinetics are complex, the annealing time between the target nucleic acid 11 and bound probes of the probe set 10 does not necessarily increase linearly as a function of length. Thus, binding time of the individual capture regions 30, 32, 34 of the interlocking probe set 10, which are respectively less than half the length of the conventional probe 53, on the target nucleic acid 11 may be significantly less than half the binding time of the conventional probe 53. Further, the specificity of the assembled interlocking probe set 10 may be similar to, or even greater than, the specificity of the conventional probe 53 to the same target binding sequence 24.

[0054] As illustrated, the combined length of nucleotides of the probe capture regions 30, 32, 34 may be about the same as or greater than the total length of the capture region of the conventional probe 53. In an embodiment, the interlocking probe set 10 may be characterized by a total combined length of its individual probe capture regions 30, 32, 34. Thus, for a pool of probe sets 10 to different targets, each individual probe set 10 may be designed to be in a range of total probe capture region length for the probe set 10 of at least 40 nucleotides, at least 50 nucleotides, at least 80 nucleotides, or at least 100 nucleotides. The capture regions among different nucleic acids of an individual probe set 10 may be of a same length or different lengths. The interlocking regions 40, 42, 44, 46 may be longer than shorter than, or about a same length as the capture regions. Different nucleic acids of an individual probe set 10 may be of a same total length or different total lengths. In an embodiment, each individual nucleic acid of the probe set, in total, is between 20-90 nucleotides in length.

[0055] In an embodiment, an individual nucleic acid 18 includes two interlocking regions 42, 44 separated by the capture region 32. Each individual interlocking region 42, 44 can be shorter than the capture region 32. However, the combined length of the interlocking 42, 44 may be longer than the capture region 32.

[0056] FIG. 2 shows an example assembly process of the individual nucleic acids 16, 18, 20 of the probe set 10 on the target nucleic acid 11. The process is initiated by contacting the probe set 10 with the target nucleic acid 11. While the illustrated process shows a single probe set specific for an individual target binding sequence 24, it should be understood that the process may occur with multiple probe sets 10 specific for different target sequences 24. If the nucleic acids 16, 18, 20 are stored together, e.g., in a single tube, some self-assembly may occur in solution. Thus, an initial step of the process may include a heating step to separate the nucleic acids 16, 18, 20.

[0057] The individual nucleic acids 16, 18, 20 can bind to the target sequence 24 independently of one another. The first nucleic acid 16 and the third nucleic acid 20 can bind, respectively to a first portion 24a and a third portion 24c of the target binding sequence 24. Thus, the binding kinetics for the nucleic acid 16 are characteristic of the length of the complementary capture region 30 to the corresponding target sequence portion 24a. Similarly, the binding kinetics for the nucleic acid 20 are characteristic of the length of the complementary capture region 34 to the corresponding target sequence portion 24c. Because these capture regions 30, 34 can be relatively shorter (e.g., 10-40 nucleotides) than the length of a conventional probe (e.g., probe 53, FIG. 1), the binding time is also shorter. In an embodiment, the individual capture regions 30, 34 bind to the target nucleic acid 11 in less than 1-3 hours, less 30 minutes or in less than 5 minutes while a conventional probe may having a target annealing time of an hour or more. As noted, the binding time may be in the context of a reaction including a plurality of different probe sets 10, each with specificity for different targets 11, and at suitable binding temperatures to avoid nonspecific binding.

[0058] Binding of the interior nucleic acid 18 between the end nucleic acids 16, 20 permits interlocking of the probe set 10 via the interlocking regions 40, 42, 44, 46. Again, the binding kinetics of the interior nucleic acid 18 binding may be according to the length of the capture region 32. Further, the interlocking kinetics may also be a function of the length of the various interlocking regions 40, 42, 44, 46. As discussed, the arrangement in FIGS. 1-2 is by way of example, and probe sets 10 with few or more nucleic acids are also contemplated, and with longer or shorter capture regions and interlocking regions.

[0059] The nucleic acids 16, 18, 20 of the probe set 10 may be bind to adjacent or contiguous target sequence portions 24a, 24b, 24c of the target sequence 24. In an embodiment, the respective target sequence portions 24a, 24b, 24c may be separated from one another by a gap of 1-5 nucleotides. That is, the nucleic acids of the probe set 10 may hybridize to noncontiguous portions of the target sequence 24 that are slightly spaced apart. [0060] The binding order of the individual nucleic acids 16, 18, 20 of the probe set 10 may be generally random and may occur in series, with any one of the nucleic acids 16, 18, 20 binding first, or at least partly in parallel. However, when at least one nucleic acid of the probe set 10 is bound to the target sequence 24, shown as nucleic acid 18 in FIG. 3, the bound nucleic acid 18 may pull down the other nucleic acids 16, 20 of the probe set 10 in a cooperative manner.

[0061] FIG. 4 is example of nonspecific binding of an individual nucleic acid (shown as nucleic acid 18) of the probe set 10 to an off-target sequence 60, which may be based on a full or partial complement between the off-target sequence 60 and the capture region 32 of the nucleic acid 18. To avoid undesired pull down of the nucleic acid fragment 62 that does not include sequences of interest, reaction or hybridization temperatures may be selected to disrupt hybridization of the individual nucleic acid 18 to the off-target sequence 60. Full probe set assembly around the off-target bound nucleic acid 18 is less likely, because other nucleic acids of the probe set 10 will not be likely to bind to the adjacent sequences 63, 64 of the off-target sequence 60. However, some interlocking via one or both of the interlocking regions 42, 44 is possible. Nonetheless, the melting temperature of the nucleic acid 18 from the off-target sequence 60 is based on the hybridized portion between the capture region 32 and the off- target sequence 60. This will be a lower temperature relative to the melting temperature of the fully assembled probe set 10 on the target binding sequence (e.g., target binding sequence 24, see FIG. 2). While other nucleic acids 16, 20 of the probe set 10 may also nonspecifically bind to off-target sequences 60, the associated nucleic acid fragment 62 is not pulled down if there is no affinity binder 50 present in the off-target nucleic acid 16, 20.

[0062] Thus, the hybridization reaction may include at least some period of time with temperatures 1) above the melting temperature associated with only binding of the nucleic acid 18 (e.g., estimated based on full or almost full complementarity as an upper bound) but 2) below the melting temperatures of the fully assembled probe set 10. In an embodiment, the reaction may start at a lower temperature (e.g., about 50-55°C) that permits early individual target binding steps in probe set assembly. Subsequently, the temperature may be increased to a temperature (e.g., about 65-75°C) above the melting temperature associated with only binding of the nucleic acid 18 (but below the melting temperature of the assembled probe set 10) to eliminate nonspecific binding when the probe sets 10 are assembled in place. In an embodiment, the lower temperature period is 1-10 minutes, and the subsequent higher temperature period is 1-60 minutes. The temperature change between the two may be a gradient or may be in stepwise changes (1-5 degree steps) with hold times of at least one minute at each step.

[0063] FIG. 5 a schematic illustration of an example assembled interlocking probe set 10 according to the disclosed embodiments. The probe set 10, as illustrated, includes five separate nucleic acids 70, 72, 74, 76, 78 that respectively bind to different part or regions of a target binding sequence 24 of the target nucleic acid 11. In the illustrated example, the affinity binder 50 is coupled to an interior-most nucleic acid 74. The nucleic acid 74 may be more stably bound to the target nucleic acid, because the flanking nucleic acids 70, 72, 76, 78 would likely be separated and uninterlocked before separation of the interior-most nucleic acid 74 from the target nucleic acid 11. In an embodiment, the lengths of the target-specific terminal regions 70, 78 can be increased relative to the interior capture regions 72, 74, 76 to provide added stability. Thus, pull down of the target nucleic acid 11, e.g., for target enrichment as shown in FIG. 7, can occur even when the probe set 10 is partially disassembled.

[0064] FIG. 6 shows partial assembly of nucleic acids on an off-target sequence 79. The nucleic acids 81, 82, 83 may be part of a probe set 10 that hybridizes to the off-target sequence 79 based on partial similarity to its target sequence 24. However, as illustrated, full assembly does not occur. Thus, in the operating temperature ranges for pull down, the nucleic acids 81 can be separated from sequence 79. This is based on the relatively lower Tm of the nucleic acids 81, 82, 83 of the partially assembled probe set 10 from the nucleic acid. Accordingly, the hybridization temperature may be selected based to disrupt nonspecific binding.

[0065] FIG. 7 shows an example target enrichment workflow using the interlocked probe sets 10 as provide herein. The term enrichment or target enrichment refers to the process of increasing the relative abundance of particular nucleic acid sequences in a sample relative to the level of nucleic acid sequences as a whole initially present in said sample before treatment. Thus the enrichment step provides a percentage or fractional increase rather than directly increasing for example, the copy number of the nucleic acid sequences of interest as amplification methods, such as PCR, would. The methods as described herein may be used to remove DNA strands that it is not desired to sequence, rather than to specifically amplify only the sequences of interest. At the level of the whole genome, removing 50% of the DNA sample gives a two-fold reduction in the cost and time of sequencing the remaining regions of biological interest from the whole genome. The methods as described herein can also be used to select large regions of a genome (e.g., megabases) for resequencing of multiple individuals, or can select out all the exons in a genomic sample. The synthesis of one array, or pool of oligonucleotides, can be used to process multiple samples of interest, and thus the costs of the oligonucleotide synthesis can be amortized over many individual samples.

[0066] The illustrated target enrichment workflow uses a panel 100 of interlocking probe sets 10 specific for respective different target nucleic acids 11. The panel 100 may include probe sets 10 having capture regions specific for a group of different target sequences 24. The target sequences targeted by the panel may include whole-exome sequencing, or predesigned or custom sequencing panels for diagnostics or screening, environmental monitoring, infectious disease surveillance, etc. Thus, each individual probe set 10 includes unique nucleic acids with sequences specific for a particular target sequence 24. Thus, the capture regions of individual nucleic acids of the probe set 10 may all be unique within the panel 100. Further, the interlocking regions of each probe set 10 may be unique to prevent a nucleic acid from a first probe set 10 from interlocking with another nucleic acid of a different probe set 10. Further, the interlocking regions are also designed to avoid hybridizing with other capture regions or target sequences of the sample.

[0067] As illustrated, the target nucleic acids may be in the form of nucleic acid fragments 102. Nucleic acid fragments 102 as provided herein, such as for target enrichment or amplification reactions may include sequence fragments that are relatively large, such as lOkb- 62Mb. In other embodiments, the fragments that are less than about Ikb in length, e.g., in the range 100-1000 base pairs in length or in the range of from 450-750 base pairs in length. It would be apparent to the skilled artisan that the following non-limiting fragmentation methods may be used: restriction endonucleases, other suitable enzymes, tagmentation viatransposases, mechanical forms of fragmentation, such as nebulisation or sonication, or non-enzymatic chemical fragmentation.

[0068] The panel 100 and fragments 102 are contacted with one another at a hybridization step 112 under conditions to permit hybridization of the probe sets 10 to their respective target nucleic acids 11, which includes assembly of the probe set 10 into the interlocked state. In an embodiment, the hybridization (e.g., binding and/or assembly of the interlocking probe set 10) to the target nucleic acid 11 as provided herein occurs at 50°C-75°C or 65°C-70°C and with hybridization times of three hours or less, two hours or less, or an hour or less to achieve desired levels of target nucleic acid binding and avoid nonspecific binding.

[0069] The hybridization step 112 may include a denaturation step in which the fragments 102 and the probe panel 100 are heated to at least 90°C (e.g., 90-95°C) to denature the fragments 102 and to separate the nucleic acids of the probe sets 10. The workflow may include a gradual or stepwise temperature decrease into the desired hybridization temperature range. In one example, after denaturation, the temperature is lowered to be at or near the melting temperature of the target capture regions of the individual nucleic acids (e.g. 50-55°C) in an uninterlocked or unassembled state. This relatively lower temperature permits binding of the individual nucleic acids of the probe set. The 50-55°C is held for a period of time (e.g., 10-20 minutes) that is relatively short. The temperature is slowly increased to the melting temperature of the fully assembled interlocked probe set (e.g. 65°C-70°C) so that any non- specifically bound probes melt off. Again, this relatively higher temperature is held for a predetermined period of time that can be relatively short (e.g., 10-20 minutes) to permit interlocking of each probe set 10 of the panel 100.

[0070] The hybridization step 112 may be performed either on the solid surface, such as on beads 120, to at least one nucleic acid of the probe sets 10 have been bound, or in solution. In certain embodiments, at least one nucleic acid of the probe set 10 may have modifications or an affinity binder 50 that facilitate separation of bound fragments 102 with an assembled probe set 10 from the unbound fragments 102. Accordingly, the probe sets 10 as provided herein may be coupled to an affinity binding molecule 50 of a binding pair, for example biotin/streptavidin, biotin/avidin, biotin/neutravidin, DNP/anti-DNP, DIG/anti-DIG, etc. and a specific antibody that binds digoxigenin are examples of specific binding pairs. In on example, biotinylation of the nucleic acid of the probe set 10 facilitates selection via streptavidin (e.g., streptavidin beads). The affinity binding molecule 50 may be an antibody ligand capable of being conjugated to a nucleotide. In certain embodiments, the modification is provided at the 5’ or the 3’ end of an individual nucleic acid of the probe set 10. Nucleic acids of the probe set 10 may also include unique barcodes or sequences (e.g., unique molecular identifiers) that facilitate identification. Such sequences may part of a region of the probe 20 that is non-complementary to the target sequence 24 and non-interlocking.

[0071] In the illustrated example, the hybridization step 112 is performed in solution, and subsequent addition of beads 120 having the mating affinity binder 122 results in binding of affinity -binder-carrying nucleic acids the probe sets 10, either as duplexes with the target sample, or as single strands. In an embodiment, the beads 120 may capture partially interlocked probe sets 10 that are not bound to a target 11. However, in one example, the hybridization temperature is selected to facilitate uncoupling of many partially assembled probe sets 10. Fragments 102 unbound to a nucleic acid of the probe set 10 that includes the affinity binder 50 will not be able to bind to the beads 120. Thus, even if individual nucleic acids of the probe set 10 are bound to the fragments 102, either at an off-target site or to the target sequence 24, capture only occurs if the affinity-binder-carrying nucleic acid is present on the fragment 102. Uncaptured fragments 102 can be removed from the beads by washing, for example.

[0072] In one embodiment the captured fragments can be removed from the probe-target complex prior to sequencing for example by elution. Removal by denaturation of the selected targets from the immobilized capture probes will generally give a solution of enriched target nucleic acid fragments 130. The enriched target nucleic acid fragments 130 can be provided for subsequent sequencing steps. In an alternative embodiment the enriched target nucleic acid fragments 130 may be amplified while still attached to the beads by, for example, emulsion phase PCR, or may be eluted from the beads and amplified in solution prior to surface attachment as part of a sequencing reaction.

[0073] In one embodiment, the fragments 102 may be fragments generated through a library preparation workflow and that include end adaptors 140 suitable for use in sequencing, e.g., that can capture the enriched target nucleic acid fragments 130 on a solid support and that can serve as primer binding sites. The adaptors 140 may be universal adaptors, e.g., that include common sequences. In an embodiment, the adaptors 140 may be Illumina sequencing adaptors (Illumina, Inc.). Accordingly, the common sequences of the adaptors 140 may tend to hybridize to one another. In such an embodiment, adaptor blockers may also be used during the hybridization step 112. The fragments may be from a single library, i.e., singleplex, or may be multiplexed from multiple libraries.

[0074] In another embodiment, the adaptors 140 may be ligated to the enriched target nucleic acid fragments 130 after elution and additional preparation steps. For example, the enriched target nucleic acid fragments 130 may also be further fragmented after elution from the beads 120 or other support. In an embodiment, it may be advantageous to capture relatively larger fragments 102, e.g., having an average size of 10 kB, and thereby require fewer probe sets 10 to select out a specific megabase region. A 10 kB region can be selected, but not easily amplified, and therefore further fragmentation, to an average of a few hundred bases may be used after the enrichment step. If a second fragmentation step is used, then the universal adaptors 140 can be ligated onto the enriched target nucleic acid fragments 130 after the elution and after the further fragmentation step.

[0075] FIGS. 8-12 refer to an amplification process in which the interlocking nucleic acids as disclosed herein may be used as forward and reverse primers of a primer pair for nucleic acid amplification. While the illustrated examples show interlocking primers for both the forward and reverse primers, it should be understood that the disclosed embodiments encompass amplification techniques in which only one of the primer pair is an interlocked primer, while the other primer is a conventional primer. Further, as illustrated, an individual amplification process may be performed with a mix of interlocked and conventional primers.

[0076] Interlocking primer sets may improve primer specificity in PCR. The interlocking structure allows for higher temperature annealing and better specificity, while still being in the optimum size range for efficient PCR. FIG. 8 shows an example forward primer set 200 amplification cycle 1. The interlocked primer set 200 has a target binding sequence 24a having a length of a capture region 208 of the locking oligo combined with a length of a capture region 210 of the forward primer 204. When the primer set 200 is assembled on the target nucleic acid 205a and interlocked, an interlocking region 210 of the forward locking oligo 202 is hybridized to a complementary interlocking region 212 of the forward primer 204. The interlocking region 212 is 5’ of the capture region 208 in the forward primer 204. The interlocking region 210 is 3’ of the capture region 206 in the locking oligo 202.

[0077] At a high annealing (e g. 65-72°C), the forward primer 204 can only anneal to the target nucleic acid 205a at this temperature if the forward locking oligo 202 is also present and locked in place. This is in contrast to conventional primer annealing in PCR, which may be set at about 5°C lower than the estimated primer melting temperature. In an embodiment, the annealing temperature may be about 72°C, which may be within optimal operating and nucleotide incorporation temperature ranges for thermostable polymerases such as taq polymerase, Pfu, Phusion, Q5. Thus, the PCR may operate more efficiently by permitting annealing at the optimal polymerase temperature. Thus, polymerase extension to generate the amplified first cycle copy 220a may occur at the annealing temperature in an embodiment. After the amplified first cycle copy 220a is generated, a denaturing step, e.g., a higher temperature of about 95°C, separates amplified first cycle copy 220a from the target nucleic acid 205a. The forward primer 204, including the interlocking region 212, is part of the amplified first cycle copy 220a, while the locking oligo 202 is not.

[0078] FIG. 9 shows the cycle 1 extension for the reverse strand target nucleic acid 205b using a reverse primer set 200b that includes a reverse locking oligo 230 and a reverse primer 232. When assembled on the target nucleic acid, the capture region 242 of the reverse primer and the capture region 244 of the locking oligo anneal to the target binding sequence 24b. The respective interlocking regions 250, 252 also bind to one another. The interlocking region 250 is 5’ of the capture region 242 in the reverse primer 232. The interlocking region 252 is 3’ of the capture region 244 in the locking oligo 230.

[0079] After the reverse primer set 200b anneals, the amplified first cycle copy 220b is generated through polymerase extension. After the amplified first cycle copy 220b is generated, a denaturing step, e.g., a higher temperature of about 95°C, separates amplified first cycle copy 220b from the target nucleic acid 205b. The reverse primer 232, including the interlocking region 250, is part of the amplified first cycle copy 220b, while the reverse locking oligo 230 is not. It should be understood that FIGS. 8-9, which show cycle 1 forward and reverse primer amplification, may occur simultaneously on respective complementary strands of target nucleic acid 205. The forward primer set 200a and the reverse primer set 200b may be part of an interlocking forward/reverse primer pair.

[0080] The locking oligos 202, 230 may have a capture region 206, 244 between about 10-40 nucleotides and an interlocking region 210, 252 between about 10-40 nucleotides. The capture region 206, 244 may be the same as, shorter than, or longer than the interlocking region 210, 252. Similarly, the forward primer 204 and/or the reverse primer 232 may have a capture region 208, 242 between about 10-40 nucleotides and an interlocking region 212, 250 between about 10-40 nucleotides. The capture region 208, 242 may be the same as, shorter than, or longer than the interlocking region 212, 250. The melting temperature of the capture regions 206, 244 from the target nucleic acid 205 may be about the same relative to one another and, individually may be higher than a melting temperature of the locking oligo interlocking regions 210, 252 with the primer interlocking regions 212, 250.

[0081] FIGS 10-11 show forward and reverse primer cycle 2 amplifications using the forward primer set 200a and the reverse primer set 200b. As shown in FIG. 10, after the second cycle polymerase extension using the forward primer set 200a, the generated second cycle copy 220c includes both primer interlocking region sequences 212, 250. Similarly, as shown in FIG. 11, after the second cycle polymerase extension using the reverse primer set 200b, the generated second cycle copy 220d includes both primer interlocking region sequences 212, 250.

[0082] As shown in FIG. 12, subsequent cycles (e.g., cycle 3 and later) could be amplified using conventional primers. In one embodiment, the conventional primers may be the forward and reverse primers 204, 232. In another embodiment, the conventional primers may be modified versions of the forward and reverse primers 204, 232 that are truncated or that include unique molecular indexes. The outer primers prime from the locking sequences 212, 250 only present on amplified copies after cycle 2. These would only anneal at lower temperatures so the PCR protocol can be set so that they take over in later cycles. That is, early cycle annealing can be at a first temperature higher than a second temperature used in later cycles. The more specific early cycle annealing at a higher temperature enriches for target amplification in the later cycles. For specificity, the concentration of the target specific and locking oligos may be lower than that of the outer primers. Locking sequences 212, 250 may include indexes or sequencing primer sequences (e.g. unique dual index primers (UDI)). Locking sequences could contain locked nucleic acids (LNAs) to improve stability.

[0083] FIGS. 8-12 show a singleplex example. However, interlocked primers can be used for multiplexed reactions as well. This could be done by designing a new locking sequence for each new primer or potentially by sharing locking sequences between primers. For example, forward primers 1 to 5 could have locking sequence A, reverse primers 1 to 5 could have locking sequence B, forward primers 6-10 could have locking sequence C, reverse primers 6- 10 could have locking sequence D, etc. That is, each individual primer set need not necessarily include unique locking sequences for each primer. Certain interlocking sequences may be shared to make primer design easier. This would be especially useful if multiplexing a large number of primers together. In another embodiment, one locking sequences can be used on all forward primers, and one locking sequence can be used on all reverse primers in a multiplexed reaction, which would simplify primer design and would make any subsequent amplification using outer primers on the locking sequences straightforward to permit universal amplification across the multiplexed strands. [0084] It should be understood that certain embodiments of the disclosed interlocked primer sets or primer pairs may be similar to those disclosed with respect to the nucleic acids of the probe sets 10, such as Tm ranges, techniques for estimating Tm, example lengths of hybridization regions (e.g., capture regions) and interlocking regions, relative positions and/or lengths of the interlocking regions and the capture regions, and binding arrangement of the locking oligo on the target nucleic acid relative to the forward or reverse primer (e g., arrangements including contiguous target binding sequences or binding arrangements in which a locking oligo binding site is spaced apart 1-5 nucleotides from the primer binding site. Further, the primer sets 200 may include additional locking oligos.

[0085] The disclosed interlocking probes and probe sets may, in embodiments, be used in conjunction with aptamer-based assays. As used herein, an aptamer may refer to a non- naturally occurring nucleic acid that has specific binding affinity for a target molecule. The binding of the aptamer to the target molecule can result in catalytically changing the target molecule, reacting with the target molecule in a way that modifies or alters the target molecule or the functional activity of the target molecule, covalently attaching to the target molecule (as in a suicide inhibitor), and facilitating the reaction between the target molecule and another molecule. In one embodiment, the target molecule is a three dimensional chemical structure, other than a polynucleotide, that binds to the aptamer through a mechanism which is predominantly independent of Watson/Crick base pairing or triple helix binding. In an embodiment, the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule.

[0086] Aptamers include nucleic acids that are identified from a candidate mixture of nucleic acids. A specific binding affinity of an aptamer for its target may refer to aptamer binding to its target generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded regions. The aptamers discussed herein can be used in any diagnostic, imaging, high throughput screening or target validation techniques or procedures or assays for which aptamers, oligonucleotides, antibodies and ligands, without limitation can be used. Aptamers as disclosed herein may be used in aptamer-based assays, such as those disclosed in U.S. Pat. Nos. 7,855,054 and 7,964,356 and U.S. Publication Nos. US/2011/0136099 and US/2012/0115752. In another embodiment, the aptamer that are captured as part of an aptamer-based assay can be detected by detection methods using interlocking probes as discussed herein. The detection results may include a notification or output of a positive or negative detection result or a relative concentration or estimated concentration for a particular aptamer ID or a particular target of the aptamer.

[0087] FIG. 13 shows an example tri-molecular assay workflow for detection of an aptamer 300 that may be used with the interlocking probe sets as provided herein. The aptamer 300 is a single-stranded nucleic acid having a fixed or substantially fixed nucleic acid sequence and that may include modified nucleotides 310 as well as a label 312. Using the conserved sequence of the aptamer 300, a probe set 10 of single- stranded oligonucleotides can be designed that include a first probe 302 (e.g., probe Hl) that hybridizes, via a region 303, to a first region 305 of the aptamer 300 (e.g., via complementary sequences) and a second probe 306 that hybridizes to a second region 307 (e.g., a different or nonoverlapping region from the first region) of the aptamer 300. The first probe 302 may include modified nucleotides 301, in an embodiment.

[0088] The second probe 306 has a hybridizing region 314 that binds directly to the second region 307 of the aptamer 300 as well as a nonhybridizing region 315 that extends from the hybridizing region 314 and does not directly bind or hybridize to the aptamer 300. The nonhybridizing region carries an identification sequence 316 that is flanked by adapter sequences 317, 318.

[0089] The workflow includes a step of contacting the aptamers 300 with the probe set 10, e.g., with the first probes 302 and the second probes 306 to form a tri-molecular complex in which the first probe 302 and the second probe 306 are hybridized to the aptamer 300. As shown, after aptamer contacting, the nonhybridizing region 314 is single-stranded. The workflow also includes a step of separating the tri-molecular complexes via a capture entity having an affinity tag binder, such as a bead 330 (e.g., a streptavidin bead) that binds to an affinity tag 332 (e.g., biotin or other affinity tags as probided herein) linked to the first probe 302. However, other arrangements are also contemplated, including column-based, flow-cell based, or substrate-based separation using a capture entity that binds to the affinity tag 332. The unbound second probes 306 are separated from the trimolecular complexes or washed away such that the PCR-amplifiable barcode, e.g., the identification sequence 316, that is specific to each aptamer 300 can be detected. After sufficient wash steps the bound second probes 306 are eluted off the beads and added into an index PCR reaction where sequencing regions including the sample specific indexes and/or other sequencing adapter sequences are incorporated.

[0090] The first probe 302 and the second probe 306 may form an interlocking probe set 10 in which the 3’ interlocking region 340 of the first probe 302 hybridizes to a 5’ interlocking region 342 of the second probe 306. The interlocking structure provides additional stability to the probe set 10 while in the tri -molecular complex. Thus, the increased stability of the interlocking probe set one or more probes 302, 306 of the probe set 10 may prevent detachment to ensure that low concentration aptamers are detected. In addition, the increased stability at higher melting temperatures may lead to decreased nonspecific binding. Thus, as provided herein, the interlocking regions of the probe set 10 may be designed to be within a desired temperature range. Thus, for an aptamer-based assay in which multiple probes sets 10 are used, each uniquely binding to a respective different aptamer 300, each probe set 10 can have a custom or tuned interlocking region to achieve a desired melting point for the probe set. FIG. 14 shows the relationship between probe melting temperature and performance in a trimolecular assay, demonstrating that shifting the melting temperature up may be associated with improved performance.

[0091] Turning back to FIG. 13, additional steps of the tri-molecular assay may include contact with primers 350, 252 that are complementary to the flanking adapter sequences 317, 318 to generate amplification products that, via amplification, have incorporated indexes or other sequences that are compatible with next generation sequencing techniques. Thus, the amplification products may form a sequencing library to be sequenced as generally provided herein to sequence the identification sequence 316 and provide a notification as part of a detection protocol.

[0092] It should be understood that the illustrated workflow may be extended to all aptamers 300 in a multiplexed aptamer-based assay in parallel. Different aptamers 300, referred to generally as aptamers 300, may have different nucleic acid sequences relative to one another, which facilitates different target specificity and different associated probe sets 10 (having different hybridizing sequences) and associated uniquely identifying identification sequences 316 associated with each individual aptamer 300. That is, each different aptamer 300 can be associated with a unique identification sequence 316.

[0093] In embodiments, the reporter probe 306 may be adapterized as part of a sequencing workflow (e.g., a target enrichment workflow as in FIG. 7). The sequencing workflow may be any suitable sequencing workflow, and the reporter probe 306 may be captured and/or modified to be compatible with the sequencing workflow. Accordingly, in embodiments, the reporter probe 306 may amplified, ligated, and/or converted to a double-stranded structure to conform with the sequencing library format. The sequencing adapters may be integral adapter sequences to the reporter probe 306. The illustrated example shows the reporter probe 306 with integral adapter sequences 317, 318. These adapter sequences may be universal Illumina® sequencing preparations, A14, B15. In other embodiments, adapters including one or more of universal capture primer sequences, barcodes, and/or sample index sequences can be incorporated into reporter probes 306 and/or oligonucleotides generated from the reporter probes 306, such as via amplification and/or ligation and extension. Certain arrangements that include indexes may incorporate a custom or bridged primer during sequencing to accommodate the different indexes.

[0094] The adapter sequences A14-ME, ME, B15-ME, ME¹, A14, B15, and ME are provided below: [0095] A14-ME: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1)

[0096] B15-ME: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2)

[0097] ME': 5'-phos-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 3)

[0098] A14: 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 4)

[0099] B15: 5'-GTCTCGTGGGCTCGG-3' (SEQ ID NO: 5)

[00100] ME: AGATGTGTATAAGAGACAG (SEQ ID NO. : 6)

[00101] The primer region or primer binding region can include a region having the sequence of a universal Illumina® capture primer or a region specifically hybridizing with a universal Illumina® capture primer. Universal Illumina® capture primers include, e.g., P5 5’- AATGATACGGCGACCACCGA-3’ ((SEQ ID NO: 7)) or P7 (5’- CAAGCAGAAGACGGCATACGA-3’ (SEQ ID NO: 8)), or fragments thereof. A region specifically hybridizing with a universal Illumina® capture primer can include, e.g., the reverse complement sequence of the Illumina® capture primer P5 ("anti-P5": 5’- TCGGTGGTCGCCGTATCATT-3’ (SEQ ID NO: 9) or P7 ("anti-P7": 5’- TCGTATGCCGTCTTCTGCTTG-3’ (SEQ ID NO: 10)), or fragments thereof.

[00102] A conserved primer region can additionally or alternatively include a region having the sequence of an Illumina® sequencing primer, or fragment thereof, or a region specifically hybridizing with an Illumina® sequencing primer, or fragment thereof. Illumina® sequencing primers include, e.g, SBS3 (5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO: 11)) or SBS8 (5’-

CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3’ (SEQ ID NO: 12)). A region specifically hybridizing with an Illumina® sequencing primer, or fragment thereof, can include, e.g., the reverse complement sequence of the Illumina® sequencing primer SBS3 ("anti-SBS3": 5’-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3’ (SEQ ID NO: 13)) or SBS8("anti-SBS8":

5’-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG-3’ (SEQ ID NO: 14)), or fragments thereof. The incorporation of sequencing primer sequences in the reporter probes 306 may be either directly or via subsequent amplification, ligation (e g., A-tailing), or other sequencing library preparation steps.

[00103] FIG. 15 shows an alternative arrangement in which the capture entity 330 binds to a 3 ’-associated affinity tag 332 of the first probe 302.

[00104] FIG. 16 shows an arrangement in which the second probe 306 carries a quencher 364 for a FRET assay to measure the dissociation of the Trimolecular complex at variable temperatures to screen for or identify suitable probe sets 10. For aptamers that already possess a label 312, e.g., a 5’ Cy3 fluorophore, a quencher 364 such as BHQ2 can be provided at 3’ end of H2. At short distances the quencher 364 would quench the fluorescence of the label 312, but once the trimolecular complex dissociates the fluorescence of the label 312 can be detected. To measure background between different probe designs equimolar aptamers may be provided as the input to the trimolecular assay and also a test with or without aptamer presence to measure aptamer independent background with different probe designs. Spike-in experiments in which a subset of aptamers is spiked into the mix. If the interlocking regions led to non-specific capture, second probe reporters would increase for not only the spiked in aptamers but other non-specific ones as well.

[00105] To decrease general background, hybridization conditions such as hybridization temperature or additives like formamide may be adjusted. Probe set design variables may include the gap length between the first probe 302 and the second probe 306 to avoid steric hindrance of interlocking regions 340, 342, interlocking region lengths and compositions and the inclusion of modifications like LNAs, and affinity tag location at the 3’ or 5’ end of first probe 302. An additional consideration is specificity such that the presence of the interlocking regions, enhances avidity without increasing background. One source of background is first probe-second probe undesired binding, and the presence of interlocking regions may increase this background. Thus, the interlocking regions may be designed with a sufficiently low enough melting temperature on their own to not hybridize to each other independent of aptamer presence. Further, mismatched first probes 302 and second probes 306 should not hybridize to each other in the trimolecular complex. To avoid this issue, the interlocking regions may have sequences between different probe sets 10.

[00106] FIG. 17 shows an example embodiment in which an interlocking probe set 10 functions to bind to both the aptamer 300 as well a reporter probe 400. The reporter probe may include features of the second probe 306, such as adapter sequences 402, 404 and an identification sequence uniquely identifying for the individual aptamer. The probe set 10 includes a first probe 410 and a second probe 420 having structural features as generally discussed herein and that interlock as generally discussed herein. An interlocking region 430 of the first probe 410 hybridizes to an interlocking region 432 of the second probe 420. The interlocking structure provides additional stability to the probe set 10 as well as the associated reporter probe 400. As noted, the increased stability at higher melting temperatures relative to arrangements without the probe set 10 may lead to decreased nonspecific binding.

[00107] The first probe 410 includes both an aptamer-binding region 436 and a reporterprobe binding region 438, and the second probe 420 includes both an aptamer-binding region 440 and a reporter-probe binding region 442. The interlock regions 430, 432 as well as the aptamer-binding regions 436, 440 and the reporter-probe binding regions 438, 442 may be uniquely associated with the aptamer 300 and/or the individual probe set 10 such that mismatches between the aptamer 300 and its reporter probe 400 are reduced. In an embodiment, the reporter-probe binding regions 438, 442 may be complementary to all or part of an identification sequence of the reporter probe 400.

[00108] In the illustrated embodiment, the probe set 10 includes an interior interlock between the interlocking regions 430, 432. The length of the first probe 410 and/or the second probe 420 may be selected for desired stability. However, the desired stability may be achieved with relatively shorter and cheaper probes lengths (shown as 40-mers by way of example).

[00109] In embodiments, the disclosed aptamer detection may occur in conjunction with dynamic range compression techniques. For example, a reaction mixture to capture the individual aptamer 300 may include the probe set 10 and the reporter probe 400. To reduce the abundancy of a high-abundancy aptamer 300 to ranges closer to average or closer to lower- abundancy aptamers 300, the reaction mixture may include one or more dummy binders. For example, the reaction may include a dummy reporter 450 that is otherwise a same sequence as the reporter probe 400 (to facilitate association with the probe set 10 and aptamer 300) but missing the end sequences having the adapters 402, 404 or having modified end sequences that are not a same sequence as the adapters 402, 404. Once bound, the dummy reporter 450 will not undergo amplification steps that rely on the sequence of the adapters 402, 404 for priming and, therefore, will not generate amplification products that are detected (e.g., via incorporation into a sequencing library). The ratio of the reporter probe 400 that generates detectable results to the dummy reporter 450 that will not be amplified and, therefore, will not be detected, can be determined based on an abundancy of the aptamer 300 of interest. In an embodiment, the dummy reporter 450 to reporter probe 400 ratio is at least 10: 1 or at least 50: 1. Other dummy structures are possible, such as dummy probe sets 10 lacking an affinity tag that are not detected and provided in a reaction with detectable affinity tagged probe sets 10.

[00110] Sequencing of the reporter probe 400 yields an identification sequence that is associated with the aptamer 300 but that is not a same sequence as the aptamer. In this manner, the aptamer identification may occur without direct sequencing of the aptamer 300 via a correlation between the aptamer 300 and its corresponding uniquely associated identification sequence of the reporter probe 400.

[00111] The disclosed techniques may include interlocking probes that can be used to detect polymorphisms. Polymorphism may refer to the occurrence in the same population of two or more alleles at one genomic locus, each with appreciable frequency. In some implementations, the alleles at the one or more polymorphism loci include single nucleotide polymorphism (SNP) alleles. In some embodiments, the method includes probe hybridization to one or more target polynucleotides of interest from the sample using an interlocking probe set as provided herein. In some embodiments, one or more of the target polynucleotides include an autosomal, Y- or X-chromosome STR. In some embodiments, one or more of the target polynucleotides include an identity-informative SNP. In some embodiments, one or more of the target polynucleotides include an ancestry-informative or a phenotype-informative SNP. In any case, sequencing results of the disclosed embodiments may include polymorphism identification based on hybridization to interlocking probes.

[00112] In one embodiment, as illustrated in FIG. 18, an interlocking probe set 480 for hybridizing to and interrogating a polymorphism in a DNA sample, is comprised of two oligonucleotide probes, a first probe 482 that is, un an embodiment, present on a surface such as a bead 483 and a second probe 484 that is not surface linked, e g., can be provided at an initial stage free in solution. The first probe 482 and second probe 484 share respective regions 492, 496 of complementarity, that in a variety of reaction conditions, for example heat or salt concentration, is not conducive to assembly via hybridization of the regions 490, 492 hybridizing to one another, However, in the presence of a target polynucleotide 500, for example, a single-stranded genomic DNA, a tripartite complex 502 can form comprising a hybridization between first probe 482 and second probe 484 and the target polynucleotide 500. In essence, the presence of the target sequence 500 promotes hybridization of the three components, in contrast to the presence of just two just of the components where no hybridization occurs. The sequences of the first probe 482 and second probe 484 are chosen such that the positions 494, 496 that are not complementary to one another, are complementary to the target 500 and such that the 3’ end of the second probe 496 abuts a polymorphic base 504 (denoted as P) under investigation. Where the tripartite complex 502 forms, a priming site is formed next to the polymorphic base 504 such that the presence of a polymerase enzyme can incorporate a fluorescently labelled nucleotide onto the 3’ end of the second probe 484 and thus identify the polymorphic base via signal generation and detection. [00113] FIGS. 19-21 illustrates an exemplary application of the concept in which a heterozygous location e g., the polymorphic base 504, is interrogated with a two colour readout system using differentially fluorescently labelled dNTPs. FIG. 19 shows an example with a sample having two different alleles 500a, 500b, with respective different nucleotides at a polymorphic base location. Contact with a probe set 480 (see FIG. 18) results in formation of two different types of tripartite complexes 502a, 502b, with either an A or G at the polymorphic base location. If the polymorphism is characterized, labelled complementary nucleotides can be provided with a polymerase to generate a readout. The readout is indicative of a mixed color as a result of some complexes 502a having a T incorporated and some complexes 502b having a C incorporated as shown in FIG. 19. FIG. 20 illustrates a similar exemplary result for a sample having only one allele, and therefore only having an A available for template during polymerase incorporation, indicating a A:A homozygous allele call at the locus. FIG. 21 illustrates another exemplary result indicating a G:G homozygous allele call at the locus. Thus, the different sample compositions provide different readouts based on the signal of the incorporated labelled nucleotide.

[00114] An example of a two colour readout assay is illustrated in FIG. 22 showing a differential hybridization assay. Differentially labelled probes 510, 512 are used to probe the genotype of a particular locus within a genome target nucleic acid 520. A matching probe 522 that contains the correct complementary base at the locus will hybridize and be identified by the colour of the fluorescent label. Mismatching probes 524 will not hybridize.

[00115] Another embodiment of the tripartite probe approach is illustrated in FIG. 23. One of the probes is provided as being otherwise a same sequence but having a different nucleotide that is complementary to a first allele (probe 530a) or a second allele (530b). Each different first probe 530a, 530b has a respective different label color. A second probe 532 has a hybridization region 534 complementary to a hybridization region 536 present on both versions of the first probe 530a, 530b. The probes assemble in the presence of a target 540, and the sequence of the target, and the identity of the allele, can be determined based on the detected color of the label. The probes 530a, 530b that can form complexes will have the complementary nucleotide at the relevant position. Thus, if only one color is detected, only one allele is present (and can be identified based on the detected color). If two alleles are present, both colors will be detected. As discussed, one of the probes 530, 532 may be coupled to a surface, e.g., bead.

[00116] In both embodiments listed above, the key benefit of the tripartite or greater plexity probe method over conventional approaches is the greater sensitivity of the method. Requiring cooperative hybridization of three or more species reduces off-target non-specific hybridization present in conventional hybridization approaches

[00117] One or more probes as discussed herein may include an affinity tag. Affinity tags can be useful for a variety of applications, for example the bulk separation of target nucleic acids hybridized to hybridization tags. As used herein, the term “affinity tag” and grammatical equivalents can refer to a component of a multi-component complex, wherein the components of the multi-component complex specifically interact with or bind to each other. For example an affinity tag can include biotin or poly-His that can bind streptavidin or nickel, respectively. Other examples of multiple-component affinity tag complexes are listed, for example, U.S. Patent Application Pub. No. 2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and Int. Patent Application Pub. No. WO 2012/061832, each of which is incorporated by reference in its entirety. In embodiments, the affinity tag capture may be antibody capture, dye capture, sequence specific binding protein dCas9, or the ME sequence associated with biotinylated Tn5.

[00118] The terms target or target nucleic acid sequence as disclosed herein may refer to nucleic acid sequences of interest that is, those which hybridize to the interlocking probes and/or primers. Thus the term includes those larger nucleic acid sequences, a sub-sequence of which binds to the probe or primer and/or to the overall bound sequence. In embodiments in which the target sequences are for use in sequencing or amplification methods, the target sequences do not need to have been previously defined to any extent, other than the bases complementary to the capture probes. [00119] The interlocking probes and/or primers hybridize to target sequences in a nucleic acid sample. The hybridization is between single stranded nucleic acid sequences. This can be achieved by a number of well-known methods in the art such as, for example using heat to denature or separate complementary strands of double stranded nucleic acids, which on cooling can hybridize to the capture probes. Where the probe sets and/or primer sets include individual nucleic acids, these may be stored together in a single vessel or may be stored separately. When stored separately, the individual nucleic acids may be provided in parallel or in series to a reaction. Further, when the probe sets are provided as part of a probe panel, the probe panel may be stored together and provide in a single reaction vessel. Alternatively, individual probe sets of the probe panel may be stored separately.

[00120] The interlocking probes and/or primers as disclosed herein are nucleic acids, such as oligonucleotides, capable of binding to a target nucleic acid sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Such probes may include natural or modified bases and may be RNA or DNA. In addition the bases in probes may be joined by a linkage other than a phosphodiester bond so long as it does not interfere with hybridization. Thus probes may also be peptide nucleic acids (PNA) in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.

[00121] The target nucleic acid can be derived from any in vivo or in vitro source, including from one or multiple cells, tissues, organs, or organisms, whether living or dead, or from any biological or environmental source (e.g., water, air, soil). For example, in some embodiments, the target nucleic acid comprises or consists of eukaryotic and/or prokaryotic dsDNA that originates or that is derived from humans, animals, plants, fungi, (e.g., molds or yeasts), bacteria, viruses, viroids, mycoplasma, or other microorganisms. In some embodiments, the target nucleic acid comprises or consists of genomic DNA, subgenomic DNA, chromosomal DNA (e.g., from an isolated chromosome or a portion of a chromosome, e.g., from one or more genes or loci from a chromosome), mitochondrial DNA, chloroplast DNA, plasmid or other episomal-derived DNA (or recombinant DNA contained therein), or double-stranded cDNA made by reverse transcription of RNA using an RNA-dependent DNA polymerase or reverse transcriptase to generate first-strand cDNA and then extending a primer annealed to the first-strand cDNA to generate dsDNA. In some embodiments, the target nucleic acid comprises multiple dsDNA molecules in or prepared from nucleic acid molecules (e.g., multiple dsDNA molecules in or prepared from genomic DNA or cDNA prepared from RNA in or from a biological (e g., cell, tissue, organ, organism) or environmental (e g., water, air, soil, saliva, sputum, urine, feces) source. In some embodiments, the target nucleic acid is from an in vitro source. For example, in some embodiments, the target nucleic acid comprises or consists of dsDNA that is prepared in vitro from single-stranded DNA (ssDNA) or from singlestranded or double-stranded RNA (e g., using methods that are well-known in the art, such as primer extension using a suitable DNA-dependent and/or RNA-dependent DNA polymerase (reverse transcriptase). In some embodiments, the target nucleic acid comprises or consists of dsDNA that is prepared from all or a portion of one or more double-stranded or single-stranded DNA or RNA molecules using any methods known in the art, including methods for: DNA or RNA amplification (e.g., PCR or reverse-transcriptase-PCR (RT-PCR), transcription- mediated amplification methods, with amplification of all or a portion of one or more nucleic acid molecules); molecular cloning of all or a portion of one or more nucleic acid molecules in a plasmid, fosmid, BAC or other vector that subsequently is replicated in a suitable host cell; or capture of one or more nucleic acid molecules by hybridization, such as by hybridization to DNA probes on an array or microarray. Target nucleic acids as provided herein may include, but are not limited to DNA, RNA, peptide nucleic acid, morpholino nucleic acid, locked nucleic acid, glycol nucleic acid, threose nucleic acid, mixtures thereof, and hybrids thereof. In an embodiment, genomic DNA fragments, or amplified copies thereof, are used as the target nucleic acid. In another embodiment, mitochondrial or chloroplast DNA is used. Still other embodiments are targeted to RNA or derivatives thereof such as mRNA or cDNA. In some embodiments, target nucleic acid can be from a single cell. In some embodiments, target nucleic acid can be from acellular body fluids, for example, plasma or sputum devoid of cells. In some embodiments, target nucleic acid can be from circulating tumor cells. [00122] The locking sequences or interlocking regions may be synthetic sequences or non- naturally occurring sequences that are not specific for any target sequences of the target source to reduce non-specific binding. Thus, the probe sets and/or primer sets as provide herein include modified nucleic acids with non-naturally occurring sequences.

[00123] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

CLAIMS What is claimed is:

1. A probe set for a target nucleic acid sequence, comprising: a first single- stranded oligonucleotide probe comprising a first probe capture region and a first probe 5’ interlocking region; a second single-stranded oligonucleotide probe comprising a second probe 3’ interlocking region complementary to the first probe 5’ interlocking region, a second probe 5’ interlocking region, and a second probe capture region; and a third single-stranded oligonucleotide probe comprising a third probe capture region and a third probe 3’ interlocking region complementary to the second probe 5’ interlocking region.

2. The probe set of claim 1, wherein the first single-stranded oligonucleotide probe is between 20-90 nucleotides in length, and wherein the first probe 5’ interlocking region complementary to the second probe 5’ interlocking region is between 10-40 nucleotides in length.

3. The probe set of claim 2, wherein the second single-stranded oligonucleotide probe is between 20-90 nucleotides in length, wherein the second probe 5’ interlocking region is between 10-40 nucleotides in length and wherein the second probe 3’ interlocking region is between 10-40 nucleotides in length.

4. The probe set of claim 3, wherein the third single-stranded oligonucleotide probe is between 20-90 nucleotides in length, and wherein the third probe 3’ interlocking region is between 10-40 nucleotides in length.

5. The probe set of claim 1, wherein the third probe capture region extends from a 5’ end of the third single-stranded oligonucleotide probe to the third probe 3’ interlocking region.

6. The probe set of claim 1, wherein the second probe capture region is flanked by the second probe 5’ interlocking region and the second probe 3’ interlocking region.

7. The probe set of claim 1, wherein the first probe capture region extends from the first probe 5’ interlocking region to a 3’ end.

8. The probe set of claim 1, wherein only the second single-stranded oligonucleotide probe carries an affinity binding molecule.

9. The probe set of claim 1, wherein the first probe capture region, the second probe capture region, and the third probe capture region are specific for nucleotides of a target nucleic acid sequence.

10. The probe set of claim 9, wherein the nucleotides are between 50-300 nucleotides in length, and wherein each of the first probe capture region, the second probe capture region, and the third probe capture region, respectively, are specific for a different subset of the nucleotides.

11. The probe set of claim 10, wherein the first probe capture region and the third probe capture region are longer than the second probe capture region or have a higher melting temperature relative to the second probe capture region.

12. The probe set of claim 11, wherein the first probe capture region, the second probe capture region, and the third probe capture region are at least 20 nucleotides in length.

13. The probe set of claim 1, wherein the first probe capture region is specific for first nucleotides of a target nucleic acid that are contiguous with or 1-3 nucleotides 5’ from second nucleotides of the target nucleic acid, wherein the second capture probe region is specific for the second nucleotides and wherein the third probe capture region is specific for third nucleotides of the target nucleic acid that are contiguous with or 1-3 nucleotides 3’ from the second nucleotides.

14. The probe set of claim 1, comprising a fourth single-stranded oligonucleotide probe comprising a fourth probe capture region and a fourth probe 3’ interlocking region complementary to a third probe 5’ interlocking region.

15. The probe set of claim 1, wherein the first probe 5’ interlocking region is shorter than the first probe capture region or has a lower melting temperature relative to the first probe capture region.

16. The probe set of claim 1, wherein one or both of the first probe capture region or the third probe capture region comprise at least one locked nucleic acid.

17. A primer composition comprising: a first single- stranded oligonucleotide comprising: a first hybridization region complementary to first nucleotides of a target nucleic acid; and a first interlocking region 3’ of the first hybridization region; and a second single-stranded oligonucleotide comprising: a second hybridization region complementary to second nucleotides of the target nucleic acid, wherein the second nucleotides are 5’ of the first nucleotides on the target nucleic acid; and a second interlocking region 5’ of the second hybridization region and complementary to the first interlocking region.

18. The primer composition of claim 17, wherein the second single- stranded oligonucleotide is a forward primer.

19. The primer composition of claim 17, wherein the second single- stranded oligonucleotide is a reverse primer.

20. A method of target enrichment, comprising: fragmenting nucleic acids of a sample to generate nucleic acid fragments comprising target sequences; contacting the nucleic acid fragments with a plurality of probes, wherein the plurality of probes comprises individual probe sets specific for respective target sequences, an individual probe set comprising: a first oligonucleotide probe comprising a first probe capture region specific for a first portion of a target sequence of the nucleic acid fragments and a first probe 5 ’interlocking region; a second oligonucleotide probe comprising a second probe 3’ interlocking region complementary to the first probe 5’ interlocking region, a second probe 5’ interlocking region, and a second probe capture region specific for a second portion of a target sequence; and a third oligonucleotide probe comprising a third probe capture region specific for a third portion of a target sequence and a third probe 3’ interlocking region complementary to a second probe 5’ region, and wherein the contacting is under conditions allowing for hybridization of the individual probe sets to the nucleic acid fragments and interlocking of the individual probe sets to form interlocked probe-fragment complexes; and separating the interlocked probe-target complexes from unhybridized nucleic acid fragments of the nucleic acid fragments to generate separated nucleic acid fragments.

21. The method of claim 20, comprising sequencing the purified nucleic acid fragments.

22. The method of claim 20, comprising heating the plurality of probes before the contacting.

23. The method of claim 20, comprising adding adaptors to ends of the nucleic acid fragments before the contacting.

24. A method of aptamer detection, comprising: contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; and detecting the analytes by detecting aptamers of the analyte-aptamer complexes, wherein detecting an individual aptamer of the plurality of aptamers comprises: contacting the individual aptamer with an interlocking probe set, wherein the interlocking probe set comprises: a first single-stranded oligonucleotide probe comprising a first complementary region that hybridizes to a first region of the individual aptamer and a first interlocking region; and a second single-stranded oligonucleotide probe comprising a second complementary region that hybridizes to a second region of the individual aptamer, a nonhybridizing region comprising an identification sequence uniquely identifying for the individual aptamer, and a second interlocking region, wherein the first interlocking region and the second interlocking region hybridize to one another; capturing a first probe of probe set via binding of the affinity tag to an affinity tag binder to capture the individual aptamer and the second probe hybridized to the second region of the individual aptamer; and detecting the identification sequence of the captured second probe.

25. The method of claim 24, wherein the first region and the second region are spaced apart by at least one nucleotide.

26. The method of claim 24, wherein the affinity tag is biotin and the affinity tag capture molecule is avidin or streptavidin.

27. The method of claim 24, wherein detecting the identification sequence of the captured second probe comprises contacting the captured second probe with primers to generate an amplification product.

28. The method of claim 27, wherein the primers comprise a first primer that binds to a first primer binding region of the nonhybridizing region and a second primer that binds to a second primer binding region of the nonhybridizing region, wherein the first primer binding region and the second primer binding region flank the identification sequence.

29. The method of claim 28, wherein the first primer comprises a first sequencing primer and the second primer comprises a second sequencing primer such that the amplification product comprises the first sequencing primer and the second sequencing primer.

30. The method of claim 27, wherein detecting the identification sequence of the captured second probe comprises sequencing the amplification product.

31. The method of claim 24, further comprising generating a notification related to the individual aptamer based on detecting the identification sequence.

32. A method of aptamer detection, comprising: contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; and detecting the analytes by detecting aptamers of the analyte-aptamer complexes, wherein detecting an individual aptamer of the plurality of aptamers comprises: contacting the individual aptamer with a reporter probe and an interlocking probe set to cause the aptamer, the interlocking probe set, and the reporter probe form a complex, wherein the interlocking probe set comprises: a first single-stranded oligonucleotide probe comprising a first aptamer complementary region that hybridizes to a first region of the individual aptamer, a first reporter probe complementary region that hybridizes to a first region of the reporter probe, and a first interlocking region; and a second single-stranded oligonucleotide probe comprising a second complementary region that hybridizes to a second region of the individual aptamer, a second reporter probe complementary region that hybridizes to a second region of the reporter probe, and a second interlocking region, wherein the reporter probe comprises an identification sequence uniquely identifying for the individual aptamer, and wherein the first interlocking region and the second interlocking region hybridize to one another in the complex; and detecting an identification sequence of the reporter probe of the complex.

33. The method of claim 32, wherein the interlocking probe set comprises an affinity tag.

34. The method of claim 33, further comprising separating the complex from unbound other reporter probes with binding activity to different aptamers using the affinity tag.

35. The method of claim 33, wherein the interlocking probe set forms a bridge between the individual aptamer and the reporter probe.

36. A method of polymorphism detection, comprising: contacting nucleic acid fragments of a target nucleic acid with a probe set specific for a polymorthe phic target sequences, probe set comprising: a first oligonucleotide probe comprising a first probe capture region specific for a first portion of the target nucleic acid, the first probe capture region terminating adjacent to a polymorphic base of the target nucleic acid and a first probe interlocking region; and a second oligonucleotide probe comprising a second probe interlocking region and a second probe capture region specific for a second portion of the target nucleic acid; extending from a 3’ end of the second probe with a labelled nucleotide using the polymorphic base as a template; and detecting incorporation of the labelled nucleotide.

37. The method of claim 36, wherein the first probe is linked to a substrate.

38. A method of polymorphism detection, comprising: contacting nucleic acid fragments of a target nucleic acid with a probe set specific for a polymorthe phic target sequences, probe set comprising: a first oligonucleotide probe comprising a first probe capture region specific for a first portion of the target nucleic acid and comprising a first base complementary to a first polymorphic base at a polymorphic location and a first probe interlocking region comprising a first label; a second oligonucleotide probe comprising the first probe capture region specific for the first portion of the target nucleic acid and comprising a second base complementary to a second polymorphic base at the polymorphic location and the first probe interlocking region comprising a second label; and a third oligonucleotide probe comprising a complent to the first probe interlocking region and a second probe capture region specific for a second portion of the target nucleic acid; and detecting one or both of the first oligonucleotide probe or the second oligonucleotide probe to characterize the target nucleic acid.