Multivalent assembly for enhancing target hybridization

Abstract

Multivalent assemblies for target hybridization are described. The multivalent assembly includes a set of oligonucleotides that hybridize to a target nucleic acid to allow capture of the target nucleic acid. In one embodiment, the multivalent assembly is heteromultivalent such that the set of oligonucleotides includes different oligonucleotides that bind to different regions of the target nucleic acid.

Description

Background Art

[0001] The subject matter discussed in this section should not be considered to be prior art simply because it is mentioned in this section. Similarly, problems mentioned in this section or associated with the subject matter provided as background technology should not be considered to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which themselves may also correspond to specific implementations of the technology protected by the claims.

[0002] The sequencing method of the next generation sequencing (NGS) platform usually adopts a nucleic acid fragment library. In the targeted sequencing technology, a subset of fragments containing the gene or region of interest of the genome is separated from the nucleic acid library and sequenced. The targeted method using NGS allows researchers to focus time, cost and data analysis on the specific area of ​​interest. Such targeted analysis can include exomes (protein coding parts of the genome), specific genes of interest (customized content), targets within genes, or mitochondrial DNA. The targeted method contrasts with the more comprehensive whole genome sequencing method, but also involves sequencing areas of the genome that may not be of interest to all users.

[0003] In one example of targeted sequencing technology, a target enrichment or hybrid pull-out method uses a batch or set of probes that hybridize to target sequences in a nucleic acid library. Hybridization of the probes to the target sequences allows these sequences to be separated from the remaining fragments in the library to enrich for targeted sequencing using the captured sequences. Summary of the invention

[0004] In one embodiment, the present disclosure provides a multivalent assembly. The multivalent assembly includes a first single-stranded oligonucleotide probe complementary to a first region of a target nucleic acid, a second single-stranded oligonucleotide probe complementary to a second region of the target nucleic acid, and a third single-stranded oligonucleotide probe complementary to a third region of the target nucleic acid. The melting temperatures of the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe from the target nucleic acid are all within 20°C.

[0005] In one embodiment, the present disclosure provides a multivalent bead assembly. The multivalent bead assembly includes a bead surface. The multivalent bead assembly also includes a first single-stranded oligonucleotide probe comprising a first hybridization sequence complementary to the first region of the target nucleic acid, a second single-stranded oligonucleotide probe comprising a second hybridization sequence complementary to the second region of the target nucleic acid, and a third single-stranded oligonucleotide probe comprising a third hybridization sequence complementary to the third region of the target nucleic acid. The first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are randomly fixed on the bead surface, and the first hybridization sequence, the second hybridization sequence, and the third hybridization sequence are different from each other.

[0006] In one embodiment, the present disclosure provides a hybridization kit. The hybridization kit includes a first bead, the first bead includes a first plurality of single-stranded oligonucleotide probes randomly fixed on the surface of the first bead, wherein a first subset of the first plurality of single-stranded oligonucleotide probes is complementary to a first region of a first target nucleic acid, a second subset of the first plurality of single-stranded oligonucleotide probes is complementary to a second region of the first target nucleic acid, and a third subset is complementary to a third region of the first target nucleic acid. The hybridization kit includes a second bead, the second bead includes a second plurality of single-stranded oligonucleotide probes randomly fixed on the surface of the second bead, wherein a first subset of the second plurality of single-stranded oligonucleotide probes is complementary to a first region of a second target nucleic acid, a second subset of the second plurality of single-stranded oligonucleotide probes is complementary to a second region of the second target nucleic acid, and a third subset of the second plurality of single-stranded oligonucleotide probes is complementary to a third region of the second target nucleic acid.

[0007] In one embodiment, the present disclosure provides a method for target enrichment. The method includes fragmenting the nucleic acid of the sample to produce nucleic acid fragments containing target nucleic acids, and contacting the nucleic acid fragments with multiple multivalent assemblies to form a multivalent assembly-target nucleic acid complex, wherein the multivalent assembly includes a separate probe set specific for the corresponding target nucleic acid. The separate probe set includes a first oligonucleotide probe complementary to the first subregion of the target nucleic acid; a second oligonucleotide probe complementary to the second subregion of the target nucleic acid; and a third oligonucleotide probe complementary to the third subregion of the target nucleic acid, wherein the first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe have sequences that can be distinguished from each other. The method also includes separating the multivalent assembly-target nucleic acid complex from the unhybridized nucleic acid fragments of the nucleic acid fragment to produce separated nucleic acid fragments.

[0008] In one embodiment, the present disclosure provides a method for cDNA synthesis. The method includes contacting an RNA sample with a multivalent assembly to capture an RNA molecule, and the multivalent assembly includes a probe set fixed on the surface of a bead. The probe set includes a first oligonucleotide probe complementary to a first subregion of the RNA molecule; a second oligonucleotide probe complementary to a second subregion of the RNA molecule; and a third oligonucleotide probe complementary to a third subregion of the RNA molecule. Wherein the first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe have sequences that can be distinguished from each other. The method also includes extending the first oligonucleotide probe using a reverse transcriptase to produce a cDNA complementary to at least a portion of the RNA molecule.

[0009] The foregoing description is presented to enable making and using the disclosed technology. 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 disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but to conform to the widest scope consistent with the principles and features disclosed herein. The scope of the disclosed technology is defined by the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] These and other features, aspects and advantages of the present invention will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters refer to like parts throughout, and in which:

[0011] Figure 1 The relationship between oligonucleotide length and total length synthesis is shown;

[0012] Figure 2 The synthesis failure for an 80-mer oligonucleotide with a hairpin structure is shown;

[0013] Figure 3 is a schematic diagram of a multivalent assembly according to aspects of the present disclosure;

[0014] Figure 4 Oligonucleotide probes of different lengths and falling within a range of melting temperatures are shown;

[0015] Figure 5 is a schematic diagram of an example multivalent assembly having oligonucleotides immobilized on the surface of a bead according to aspects of the present disclosure;

[0016] Figure 6 is a schematic diagram of an example multivalent assembly with oligonucleotides immobilized on beads according to aspects of the present disclosure;

[0017] Figure 7 is a schematic diagram of an example surface of a multivalent assembly with randomly seeded oligonucleotides according to aspects of the present disclosure;

[0018] Figure 8 is a schematic diagram of an example multivalent assembly including a nucleic acid tile scaffold according to aspects of the present disclosure;

[0019] Fig. 9 is a schematic diagram of an exemplary multivalent assembly including a polypeptide scaffold according to aspects of the present disclosure;

[0020] Fig.10 An example target enrichment workflow using a multivalent assembly according to aspects of the present disclosure is shown;

[0021] Fig.11An example cell-free nucleic acid capture using a multivalent assembly according to aspects of the present disclosure is shown;

[0022] Fig.12 shows an example nucleic acid capture using multivalent assemblies with random N-mers according to aspects of the present disclosure;

[0023] Fig.13 An example nucleic acid capture using a multivalent assembly with targeting oligonucleotides according to aspects of the present disclosure is shown;

[0024] Fig.14 An example cDNA extension using a multivalent assembly according to aspects of the present disclosure is shown;

[0025] Fig.15 The performance of hybridization-based extraction was shown to exceed that of total nucleic acid extraction;

[0026] Fig.16A The experimental workflow for measuring the use of melting temperature as a proxy for increased affinity is shown;

[0027] Fig. 16B It shows that for Fig.16A The fluorescently labeled targets in the image are combined with the images of the results according to the temperature reduction;

[0028] Fig.17A Example 1 probe beads, Example 3 probe beads, and Example 5 probe beads are shown;

[0029] Fig. 17B The melting temperatures for 1-probe beads, 3-probe beads, and 5-probe beads are shown, showing the binding coordination of the multivalent assembly to different probe types;

[0030] Fig.18 An enrichment scheme suitable for bead enrichment is shown;

[0031] Fig.19A Exemplary heteropolyvalent assemblies and monovalent assemblies are shown;

[0032] Fig.19B Comparison of target enrichment between heteropolyvalent and monovalent assemblies is shown;

[0033] Fig.19C Comparison of target enrichment between heteropolyvalent and monovalent assemblies is shown;

[0034] Fig. 20A Multivalent bead read enrichment for the forward and reverse strands is shown; and

[0035] Fig. 20B The lambda phage library workflow for probes in solution against multivalent bead pairs is shown. DETAILED DESCRIPTION

[0036] The following discussion is presented to enable any person skilled in the art to make and use the disclosed technology, and is provided in the context of a specific application and its requirements. Various modifications to the disclosed implementations will be 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 disclosed technology. Therefore, the disclosed technology is not intended to be limited to the specific implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0037] The use of nucleic acids specific to the target sequence allows for target enrichment, amplification, purification, extension or other reactions. Hybridization probe capture of a subset of nucleic acid sequences from a biological sample or from a library modified with a sequencing adapter is used for enrichment and array-based expression profiles in NGS, and for applications such as pathogen detection and disease monitoring. Determination is designed with oligonucleotide probes or binding agents that have high binding affinity and high specificity for the desired analyte relative to other oligonucleotide sequences. Enrichment techniques can employ a workflow that includes hybridization of a biotinylated probe with a nucleic acid sequence of interest, pull-down of the biotinylated probe on streptavidin-functionalized magnetic beads, washing of beads to remove non-specifically bound molecules, elution of the target nucleic acid from the beads, and sequencing.

[0038] The relatively long nucleic acids in these assays can provide the desired target specificity relative to shorter nucleic acids and reduce the incidence of off-target binding. The binding strength or affinity of a nucleic acid to a target increases with the number of complementary nucleotides. However, the synthesis of longer nucleic acids for specific binding reactions is relatively more expensive and more complicated. Figure 1 The relationship between oligonucleotide length and the percentage of manufactured products representing full-length oligonucleotides is shown. As oligonucleotide length increases, the manufacturing yield of the desired full-length product decreases. In some cases, the continuous enrichment probe can be about 80 to 120 nucleotides. For high-throughput oligonucleotide synthesis, the stepwise yield can be in the range of 88% to 90%, and for oligonucleotides with a length of 80 to 120 bases with minimal secondary structure, the final product yield is 15% to 30%. Figure 2 As shown, with a higher T m The synthesis yield of oligonucleotides with secondary structures such as G-quadruplexes is poor.

[0039] In manufacturing, only full-length probes are selected for determination. Therefore, most of the synthetic products are lost, which makes the manufacture of oligonucleotides with 80 to 120 nucleotides less efficient relative to shorter oligonucleotides. Low probe manufacturing efficiency may cause additional challenges in the application of workflow with high-level excessive oligonucleotide probes relative to sample input. Hybridization capture enrichment applications can use 200ng to 500ng of high library input (which can then be related to preamplification steps) and 2000X molar excess of high oligonucleotide probe concentration to achieve desired target enrichment. Even if excessive probe concentration is used, due to the high temperature secondary structure in these longer probes, there is variable enrichment, which causes poor synthesis and poor capture of target region. Therefore, it may be challenging to produce enough probe concentrations to realize high-level excessive probes in some workflows, particularly for the probe length associated with low manufacturing yield.

[0040] Provided herein are multivalent assemblies, nucleic acids, reagents, test kits, probe groups, and methods for making and using multivalent assemblies.Multivalent assemblies can be a part of an isolation reaction, or can be used as a part of a larger workflow (such as a sequencing workflow).Disclosed technology provides coordinated shorter oligonucleotides, which avoid low target affinity and have an improved synthesis yield compared to relatively long probes.Compared to a single long probe, a multivalent assembly using multiple shorter probes of the same general region of a target nucleic acid is provided.Present technology provides a collaborative binding oligonucleotide set (e.g., a probe set), which includes these split or shorter oligonucleotides that are bonded to different regions of a target nucleic acid.In one embodiment, the target region of a target nucleic acid is composed of the shorter subregions of these independent oligonucleotide targets.This coordination increases hybridization strength (avidity) while having the possibility of reducing probe and input requirements, increases the stringency of washing for nonspecific dissociation, and avoids the potential secondary structure associated with relatively long probes, which relatively long probes cause lower manufacturing efficiency and poor target capture.

[0041] In an embodiment, oligonucleotide can be used together with multivalent surface, heteropolyvalent surface (for example, pearl) or branched oligonucleotide, each of which solves the problem of inefficient, probe-based capture of nucleic acid.Multivalent assembly includes a group of unique oligonucleotide sequences, and this group of oligonucleotide sequences can each bind to the different extensions of nucleic acid region.The combination of the independent unique oligonucleotide sequences of multivalent probe structure (such as pearl, branched oligonucleotide or oligonucleotide handle) coordination, these oligonucleotide sequences can each be attached to the different extensions of target.This coordination hybridization increases the affinity of multivalent structure to its target, without the need to increase the length of each independent probe.Enhanced affinity can also increase hybridization kinetics, to allow faster annealing time relative to longer probe, and reduce required probe concentration and input in hybridization reaction.In an embodiment, variable length probe is used in multivalent assembly to avoid secondary structure and improve the uniformity of hybridization intensity.

[0042] In an embodiment, the cooperative binding oligonucleotide using the set generally provides a binding specificity to the target nucleic acid that is suitable for a single continuous probe in the same region across the target nucleic acid. Therefore, the manufacturing complexity associated with a longer probe can be avoided while maintaining avidity. In addition, the present technology can use an assembly hybridized with two chains from a nucleic acid duplex but uses physical forward and reverse complementary probes (for example, separated on an independent bead) to achieve more effective probe capture. Probe targeting two chains in the solution is more challenging because the hybridization capture of the target region with forward and reverse complementary probes in the reaction solution causes probe-probe hybridization and reduction. In order to avoid the probe-probe hybridization of forward strand probe and reverse strand probe, some technology only targets a chain in the two chains, which makes hybridization capture challenging to the library with lower input and no PCR. The disclosed technology can avoid undesirable probe-probe hybridization while promoting the targeting of two double chains. The disclosed technology avoids the low probe synthesis efficiency for longer lengths or problematic sequences by decoupling the probe sequence with the fixed probe in some arrangements. That is, by splitting the probe into smaller fragments, synthetic complexity is avoided, but similar hybridization efficiency can be achieved using cooperative binding. In embodiments, the disclosed technology provides enhancement of on-target enrichment compared to sequential oligonucleotide hybridization capture methods as shown using the lambda genome as a model system.

[0043] Figure 31 is a schematic diagram of a multivalent assembly 10 in which a collection 12 of oligonucleotides 20 (e.g., oligonucleotide probes) having a distinguishable sequence and having binding specificity to a target nucleic acid 14 is immobilized on a surface 34. The oligonucleotide collection 12 includes individual single-stranded oligonucleotides 20 (e.g., single-stranded oligonucleotide probes), each of which has complementarity to a different region of a target nucleic acid 14. In one embodiment, the collection 12 is an oligonucleotide probe collection. As provided herein, an oligonucleotide 20 can refer to an oligonucleotide probe or a probe for a target enrichment workflow.

[0044] As provided herein, multivalent assembly 10 provides multiple hybridization sites or binding sites for target (such as target nucleic acid 14).Therefore, in one embodiment, multivalence refers to the structure with more than one hybridization site or binding site.Individual hybridization site can include separate individual oligonucleotide 20 separated from each other.Therefore, in the illustrated embodiment, the individual hybridization site shown as A', B', C', D' and N' is non-continuous and is not located on the same oligonucleotide chain.That is, in one embodiment, each individual oligonucleotide 20 including the hybridization site complementary to the region of target nucleic acid 14 has corresponding 5' end and 3' end.Therefore, as provided herein, independent oligonucleotide 22,24,26,28 and 30 include different individual hybridization sites A', B', C', D' and N'.As shown in the figure, assembly 10 is heteropolyvalent, so that oligonucleotide 20 has distinguishable or unique nucleotide sequence relative to other oligonucleotides 20 in set 12.But, other multivalent arrangements can also be expected. For example, one or more oligonucleotides of the individual oligonucleotides 20 of the set 12 may include an identical sequence relative to the other oligonucleotides of the set 12 .

[0045] The individual sets 12 include a plurality of independent oligonucleotides 20 that are complementary to different regions of the target sequence 14, respectively. In the illustrated example, oligonucleotides 22, 24, 26, and 28 (with hybridization sites A', B', C', D', and N') represent different oligonucleotides 20 of the set 12. Oligonucleotide 30 represents one or more additional oligonucleotides. Oligonucleotides 22, 24, 26, 28, and 30 are complementary to target binding regions 23, 25, 27, 29, and 31 (also represented as regions A, B, C, D, and N) of the target nucleic acid 14, respectively. The target binding regions represent different subregions of the target nucleic acid 14. In one embodiment, the target nucleic acid 14 can be a single-stranded nucleic acid or a nucleic acid fragment, and the target binding region includes only a portion of the fragment in total. That is, the target nucleic acid 14 may include a non-hybridized region that is not complementary to the oligonucleotide 20. However, binding to the oligonucleotide 20 allows the capture and enrichment of the bound fragments, including any non-hybridized regions.

[0046] In one embodiment, the length of each oligonucleotide 20 can be between 10 and 80 nucleotides. By way of example, the length of the independent oligonucleotide 20 of set 12 can be 10 to 20 nucleotides, 10 to 30 nucleotides, 20 to 30 nucleotides, 10 to 50 nucleotides or 30 to 50 nucleotides. The complementary target binding region on target nucleic acid 14 is relatively shorter (for example, 10 to 80 nucleotides in one embodiment) than the binding region (its length can be 100 to 300 nucleotides) of conventional continuous nucleic acid hybridization probe or single nucleic acid hybridization probe. However, in general, the span or length across all target binding regions 23, 25, 27, 29 and 31 can be for example at least 80 nucleotides, 80 to 150 nucleotides, 80 to 200 nucleotides, 100 to 300 nucleotides or 120 to 300 nucleotides. In one embodiment, the total length of the oligonucleotide 20 extending from 5' end to 3' end is complementary to the corresponding target binding region. In other embodiments, one or more of the oligonucleotides in oligonucleotides 20 of set 12 may include a non-hybridizing region at the 5' end and / or the 3' end.

[0047] In one embodiment, oligonucleotide 20 is complementary to the region or continuous region directly adjacent. For example, target binding region 23, 25, 27, 29 and 31 can form a continuous span or extension of target nucleic acid 14. In other embodiments, the spacer region may be present between one or more of target binding region 23, 25, 27, 29 and 31. By way of example, the length of the spacer region can be 1 to 10 nucleotides (for example, 1 to 4 nucleotides, 1 to 5 nucleotides). In other examples, the spacer region can be longer to accommodate different multivalent assembly arrangements. For example, a polypeptide base or DNA tile surface 34 can produce an oligonucleotide that binds to a target binding region, and these target binding regions are spaced 10 to 25 or more nucleotides between at least two target binding regions in the target binding region. In one embodiment, target binding region 23, 25, 27, 29 and 31 are non-overlapping and arranged in the order of 5' to 3', or vice versa.

[0048] In the illustrated example, the oligonucleotide set 12 includes at least four independent oligonucleotides 20. However, it should be understood that more or less may be included in the set 12 as generally discussed herein. The number of unique oligonucleotides 20 of the set 12 may depend on variables including the size and shape of the surface 34 and the length of the optional spacer region. For example, in one embodiment, the oligonucleotide set 12 includes a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide.

[0049] It should be understood that in embodiments, the multivalent assembly 10 can be provided with the oligonucleotides 20 in a single-stranded state (e.g., a non-hybridized state or an unbound state). However, if a target nucleic acid 14 having a sequence complementary to some or all of the oligonucleotides 20 in the set 12 is present under favorable hybridization conditions (e.g., temperature), the complementary oligonucleotides 20 can hybridize with the target nucleic acid 14. Thus, when hybridized with the target nucleic acid 14, the oligonucleotides 20 are in a duplex state or a double-stranded state.

[0050] Surface 34 may be a bead 50 (see Figure 5 ), such as magnetic beads. In embodiments, surface 34 can be a substrate, a flow cell surface, a flat surface, a shaped surface, a porous surface, a patterned surface, or a molecule such as a DNA tile structure (see Figure 8 ) or polypeptide (see Fig. 9 ).

[0051] Target binding for set 12 includes multiple shorter binding interactions with correspondingly shorter respective target binding regions, which means that multivalent assembly 10 has binding kinetics that are more highly similar to the kinetics of shorter nucleotides. In one embodiment, multivalent assembly 10 can achieve target specificity at or close to that of conventional probes with improved binding kinetics of shorter nucleic acids.

[0052] As provided herein, a probe set 12 or multivalent assembly 10 can include two or more oligonucleotides 20, whereby each individual oligonucleotide 20 of the set 12 is complementary to a corresponding portion of a target nucleic acid 14 (e.g., a single-stranded target nucleic acid 14). However, within a particular set 12, the corresponding oligonucleotides 20 can have non-uniform lengths or different lengths (e.g., different nucleotide lengths). The corresponding lengths of the oligonucleotides 20 can be selected to achieve a desired melting temperature (Tm) or within a specific melting temperature range.

[0053] Figure 4Shown are example groups of oligonucleotides with variable length but still with melting temperatures within a specific Tm range, so that the difference between the lowest Tm and the highest Tm of set 12 is less than or equal to a predetermined value. In one embodiment, the melting temperatures of set 12 are within the range of twenty degrees Celsius or ten degrees Celsius relative to each other. In one embodiment, the oligonucleotide length of set 12 can be variable, but also within a predetermined length range and with a melting temperature within a specific Tm range. In one embodiment, the longest oligonucleotide 20 of separate set 12 can be 5 to 15 nucleotides or 5 to 10 nucleotides longer than the shortest oligonucleotide 20 of separate set 12. In one embodiment, the length of the oligonucleotide 20 of separate set 12 can be between 20 and 30 nucleotides. In one embodiment, at least one oligonucleotide 20 of separate set 12 has different lengths relative to other oligonucleotides 20 of set 12. In one embodiment, each oligonucleotide 20 of separate set 12 has different lengths relative to other oligonucleotides 20 of set 12.

[0054] In one embodiment, the oligonucleotides 20 of the individual sets 12 have similar Tms for melting or dissociating from the target nucleic acid 14 relative to each other. In addition, in embodiments where a plurality of different probe sets 12 are used (see Fig.10 ), all different sets 12 can be designed so that all or most of the oligonucleotides in the oligonucleotides 20 of each different probe set 12 fall within a preset estimated Tm range. In one embodiment, the estimated Tm range of the oligonucleotides is selected between 50°C and 70°C or between 55°C and 65°C. In one embodiment, the estimated Tm range of the oligonucleotides is selected so that all nucleotides in the set 12 have a melting temperature within a temperature range of 20 degrees Celsius relative to each other. In one embodiment, the estimated Tm range of the oligonucleotides is selected so that all nucleotides in the set 12 have a melting temperature within a temperature range of ten degrees Celsius relative to each other. In one embodiment, the estimated Tm range of the oligonucleotides is selected so that all nucleotides in the set 12 are within 5 degrees Celsius to 20 degrees Celsius, within 10 degrees Celsius to 20 degrees Celsius, or within 15 degrees Celsius to 20 degrees Celsius. By making the probes have different lengths or more variable lengths, a more uniform Tm between the probes can be achieved. Additionally, certain multivalent assemblies 10 may include modified nucleic acids (eg, locked nucleic acids) to enhance stability and hybridization with target nucleic acids 14 .

[0055] In one embodiment, the Tm of a particular individual oligonucleotide 20 or fully assembled multivalent assembly 10 for a probe set 12 can be estimated based on the following assumed nearest neighbor formula:

[0056]

[0057] in:

[0058] T m = melting temperature in °C

[0059] ΔH = kcal mol -1 Enthalpy change (considering the energy change during annealing / melting)

[0060] A = constant -0.0108 kcal K -1 ·mol -1 (Consider helix initiation during annealing / melting)

[0061] ΔS = K in kcal -1 ·mol -1 The entropy change (considering the energy that cannot do work, i.e. disorder)

[0062] R = gas constant 0.00199 kcal K -1 ·mol -1 (Constant for converting energy to temperature)

[0063] C = in M ​​or mol L -1 The oligonucleotide concentration calculated (Table 1 shows 0.0000005, i.e. 0.5 μM)

[0064] -273.15 = Conversion factor to change expected temperature in Kelvin to °C

[0065] [Na + ] = in M ​​or mol L -1 The sodium ion concentration (Table 1 shows 0.05, i.e. 50 mM)

[0066] Table 1: Changes in enthalpy and entropy

[0067]

[0068] Table 1: *Left sequence is 5' to 3', while right sequence is 3' to 5', for example, for AA / TT, AA is 5' to 3 and TT is 3' to 5'. When selecting a value, always select in the 5' to 3' direction, regardless of whether it is a 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 result in the same T m calculate.

[0069] As another example, Tm can be estimated as follows:

[0070] ●For sequences with less than 14 nucleotides, the formula is:

[0071] Tm=(wA+xT)*2+(yG+zC)*4

[0072] Where w, x, y, z are the number of bases A, T, G, and C in the sequence, respectively.

[0073] ●For sequences longer than 13 nucleotides, the equation used is:

[0074] Tm=64.9+41*(yG+zC-16.4) / (wA+xT+yG+zC)

[0075] The above equation assumes annealing at 50 nM primer, 50 mM Na + and pH 7.0. In one embodiment, the sequence length is based on the total length of the nucleic acid.

[0076] Figure 5 An example multivalent assembly 10 is shown in which a collection 12 of oligonucleotides 20 is immobilized on a surface 34 of a bead 50. The illustrated example is a heteropolyvalent arrangement in which multiple oligonucleotides having different sequences are immobilized on a single particle (e.g., a magnetic bead). The number of unique probes per particle can be empirical but depends on variables including the size of the particle and the length of the optional linker 36, such as Figure 6 As shown. The connector 36 provides flexibility and length away from the beads 50 so that the probes can be arranged in the correct order to coordinate hybridization. The connector 36 can be a universal connector 36, which is the same connector even for different oligonucleotides. The connector 36 can be a nucleotide connector, a chemical connector, or a polymer connector. In one embodiment, the disclosed multivalent assembly can include a hybridization probe set, which can be used, for example, for target enrichment NGS. For hybrid capture, many particle types or multivalent assemblies (each with their own probe set 12) are combined together to enrich the desired group of target areas.

[0077] like Figure 7 As shown, the surface 34 of the beads or other structures can be randomly seeded with oligonucleotides 20 so that subsets 56 of oligonucleotides 20 are arranged in the correct order relative to each other to promote hybridization with the target nucleic acid 14. The illustrated embodiment shows a single subset 56. However, depending on the surface density of the oligonucleotides 20, multiple subsets 56 can be formed on the surface 34. Therefore, each bead 50 or other assembly 10 can be capable of hybridizing multiple targets if available in a given sample. In one embodiment, each set 12 of oligonucleotides 20 can include multiple copies of each individual oligonucleotide 20 representing different heterovalent binding sequences.

[0078] exist Figure 8In the example multivalent assembly 10, oligonucleotides 20 are fixed on pre-folded DNA tiles 60. DNA origami technology is used to assemble tile bases with variable hook arms 64 of different nucleotide sequences, which can be used to hybridize with linker sequences 66 coupled to the oligonucleotides 20. This allows a single universal tile base 60 to be used for many different sets 12 so as to capture a variety of different targets in a combined manner. In addition, the hooks 64 can be arranged so that the oligonucleotides 20 are arranged in sequence to hybridize with the target according to their sequences. For hybridization capture, many tiles (each with their own probe set 12) are combined together to enrich the desired group of target regions. The tile 60 is coupled to an affinity molecule 68 (such as biotin), which is bound to an affinity molecule binder (such as streptavidin) to facilitate the separation of the bound target nucleic acid 14 from other components of the sample.

[0079] Fig. 9 An example multivalent assembly including a polypeptide scaffold 70 is shown. Oligonucleotides 20 can be attached to a universal peptide, such as a polypeptide scaffold 70, in an array manner through multiple orthogonal chemistries. Peptide synthesis of alpha helices with several orthogonal amino acid chemical handles can be used to sequentially attach oligonucleotides 20. Oligonucleotides 20 can be arranged according to the target sequence to allow target capture. Coupling to affinity molecules 68 allows separation after hybridization. Several peptide: oligonucleotide multivalent assemblies 10 specific for corresponding different target nucleic acids 14 (e.g., having different sequences) can be combined for parallel enrichment on the desired target area.

[0080] The multivalent assembly 10 may include oligonucleotides 20 covalently attached to a collection 12 of biotinylated scaffolds (such as dendrimers or bottlebrushes) at multiple branch points. These multivalent assemblies 10 hybridize with their targets in solution and can be captured on streptavidin beads. In the bottlebrush method, terminal deoxynucleotidyl transferase either extends azide-linked nucleotides from ssDNA oligonucleotides, or an alternative DNA polymerase adds azide-linked nucleotides by amplification of the template. After the azide-linked nucleotides are incorporated, the oligonucleotides 20 are covalently attached by azide-alkyne cycloaddition (click chemistry). Coupled to a branched arrangement with a more flexible scaffold relative to the beads can lead to better coordination of the probes with their respective targets and also avoid sedimentation or agglomeration.

[0081] Fig.10An example target enrichment workflow using a multivalent assembly 10 as provided herein is shown. The term "enrichment" or "target enrichment" refers to a process of increasing the relative abundance of a specific nucleic acid sequence in the sample relative to the level of the nucleic acid sequence that was initially present in the sample as a whole before processing. Therefore, the enrichment step provides a percentage or fractional increase, rather than directly increasing the copy number of the nucleic acid sequence of interest as an amplification method (such as PCR). The methods described herein can be used to remove DNA chains that are not expected to be sequenced, rather than only specifically amplifying sequences of interest. At the level of the whole genome, removing 50% of the DNA sample reduces the cost and time of the remaining biological region of interest from whole genome sequencing by two times. The methods described herein can also be used to select large regions of the genome (e.g., megabases) for re-sequencing of multiple individuals, or all exons in a genomic sample can be selected. The synthesis of an array or library of oligonucleotides can be used to process multiple samples of interest, so the cost of oligonucleotide synthesis can be apportioned to many individual samples.

[0082] The illustrated target enrichment workflow uses a group 100 of multivalent assemblies 10 specific for respective different target nucleic acids 14. The group 100 may include multivalent assemblies 10 capable of hybridizing with different target sequences of a selected group. The target sequences targeted by the group may include whole exome sequencing, or pre-designed or customized sequencing groups for diagnosis or screening, environmental monitoring, infectious disease monitoring, etc. Thus, each multivalent assembly includes a collection 12 of unique oligonucleotides 20 having a sequence specific to a particular target sequence 14. Thus, the hybridization sequences of the individual oligonucleotides 20 of the collection 12 may all be unique within the group 100. In the illustrated example, the oligonucleotides 20 may be used as hybridization probes.

[0083] As shown, target nucleic acid 14 can be in the form of nucleic acid fragment 102. As provided herein, nucleic acid fragment 102 (such as for target enrichment or amplification reaction) can include relatively large sequence fragments, such as 10 kilobases (kb) to 62 megabases (Mb) in length. In other embodiments, the fragment length is less than about 1kb, for example, the length is within the range of 100 to 1000 bases or the length is within the range of 450 to 750 bases. It will be apparent to those skilled in the art that the following non-restrictive fragmentation methods can be used: restriction endonucleases, other suitable enzymes, fragmentation of the enzyme digestion method carried out via transposase, fragmentation of mechanical forms (such as atomization or ultrasonic treatment) or non-enzymatic chemical fragmentation.

[0084] The group 100 and the fragments 102 are contacted with each other at a hybridization step 112 under conditions that allow the oligonucleotides 20 of the multivalent assembly 10 to hybridize with their corresponding target nucleic acids 14. The hybridization results in the formation of a multivalent assembly-target nucleic acid complex 120 for at least some of the fragments in the fragments 102 and at least some of the multivalent assemblies in the multivalent assembly 10. That is, hybridization occurs when the target nucleic acid 14 is present in the fragments 102. In addition, some of the fragments 102 may not have any sequence that is a target of the group 100. In one embodiment, hybridization with the target nucleic acid 14 as provided herein (e.g., binding and / or assembly of the set 12) is performed at 50° C. to 65° C., and the hybridization time is three hours or less, two hours or less, or one hour or less, to achieve the desired level of target nucleic acid binding and avoid nonspecific binding.

[0085] Hybridization step 112 may include a denaturation step, wherein fragment 102 and probe group 100 are heated to at least 90°C (e.g., 90°C to 95°C) to denature fragment 102 and separate the nucleic acid of multivalent assembly 10. Workflow may include gradually or stepwise lowering the temperature to a desired hybridization temperature range. In one example, after denaturation, the temperature is lowered to a melting temperature (e.g., 50°C to 65°C) equal to or lower than the melting temperature of a single oligonucleotide. This relatively low temperature allows the combination of a single oligonucleotide 20 of set 12. Keep 50°C to 65°C for a relatively short time period (e.g., 10 minutes to 20 minutes). Slowly raise the temperature to allow any non-specifically bound probe to melt. Similarly, this relatively high temperature is kept for a relatively short predetermined time period (e.g., 10 minutes to 20 minutes).

[0086] The hybridization step 112 can be performed on a solid surface 34 (such as on a bead 50), or in a solution. In certain embodiments, at least one nucleic acid of the multivalent assembly 10 may have a modification or affinity binding agent 68 that promotes the separation of the bound fragment 102 from the unbound fragment 102. Therefore, the multivalent assembly 10 as provided herein can be coupled to the affinity binding molecule 68 of the binding pair, such as biotin / streptavidin, biotin / avidin, biotin / neutravidin, DNP / anti-DNP, DIG / anti-DIG, etc., and the specific antibody that binds digoxin is an example of a specific binding pair. In one example, the biotinylation of the nucleic acid of the multivalent assembly 10 promotes the selection via streptavidin (e.g., streptavidin beads). The affinity binding molecule 68 can be an antibody ligand that can be conjugated to a nucleotide. In certain embodiments, a modification is provided at the 5' end or 3' end of a single nucleic acid of the multivalent assembly 10. The nucleic acid of the multivalent assembly 10 may also include a unique barcode or sequence (e.g., a unique molecular identifier) ​​that facilitates identification. In embodiments where the multivalent assembly 10 includes beads 50, the beads 50 may be magnetic beads that can be separated using magnetic pull-down. In other embodiments, the beads 50 may include an affinity binder 68.

[0087] In certain embodiments, the hybridization step 112 can be performed in solution, and the subsequent addition of beads with paired affinity binders results in the binding of the multivalent assembly 10 carrying the affinity binder as a duplex with the fragment or as a single strand. For example, when the multivalent assembly 10 includes a DNA or polypeptide scaffold coupled to the affinity binder. The multivalent assembly 10 can be hybridized in solution with a fragment 102 comprising a target sequence, and the multivalent assembly 10 can be captured via the affinity binder. For example, the uncaptured fragment 102 can be removed from the beads by washing.

[0088] In one embodiment, the captured fragment can be removed from the probe-target complex before sequencing, for example, by elution. Removing the selected target from the fixed capture probe by denaturation will generally result in a solution of enriched target nucleic acid fragments 130. Enriched target nucleic acid fragments 130 can be provided for subsequent sequencing steps. In an alternative embodiment, the enriched target nucleic acid fragments 130 can be amplified by, for example, emulsion phase PCR while still attached to the beads, or can be eluted from the beads and amplified in solution as a part of sequencing reactions before surface attachment.

[0089] In one embodiment, fragment 102 can be a fragment produced by a library preparation workflow, and it includes a terminal adapter 140 suitable for sequencing, for example, it can capture the enriched target nucleic acid fragment 130 on a solid support and can serve as a primer binding site. Adapter 140 can be a universal adapter, for example, a universal adapter including a public sequence. In one embodiment, adapter 140 can be an Illumina sequencing adapter (Illumina, Inc.). Therefore, the public sequences of adapter 140 can tend to hybridize with each other. In such embodiments, adapter blockers can also be used during hybridization step 112. Fragments can come from a single library (i.e., a single body) or can be multiplexed from multiple libraries.

[0090] In another embodiment, the adapter 140 can 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 can also be further fragmented after elution from the beads 120 or other carrier. In one embodiment, it may be advantageous to capture relatively large fragments 102, for example having an average size of 10 kB, and thereby require fewer probe sets 12 to select specific megabase regions. 10 kB regions can be selected, but are not easily amplified, and therefore further fragmentation of an average of several hundred bases can be used after the enrichment step. If a second fragmentation step is used, the universal adapter 140 can be ligated to the enriched target nucleic acid fragments 130 after elution and after a further fragmentation step.

[0091] In another embodiment, the disclosed multivalent assembly 10 can be used for nucleic acid capture. Fig.11 An example of capturing cell-free DNA and / or RNA from a biological sample via a multivalent assembly 10 based on beads is shown. The use of multivalent beads or other multivalent assemblies 10 to capture nucleic acids from a biological sample provides the advantages of traditional purification methods (such as purification based on columns). The purification and enrichment of multivalent assemblies 10 coupled target fragments. For example, probes can be used to deplete unwanted nucleic acids from a sample, such as abundant RNA, such as ribosomal RNA. Compared with traditional methods, multivalent assemblies 10 can capture target fragments with higher sensitivity at a lower copy number due to their high affinity. Once nucleic acids are captured on the multivalent assembly 10, the captured nucleic acids can be enzymatically manipulated to add sequences (such as sequencing adapters) on the bead surface. This will enable enzymes and buffers to be exchanged while nucleic acids are coupled to beads.

[0092] The multivalent assembly 10 can be used to convert RNA to cDNA on beads (ie, cDNA conversion). Figure 12 to Figure 13Capturing nucleic acids via multivalent beads to convert RNA into cDNA libraries is described. Traditional cDNA synthesis uses hexamers to convert large amounts of RNA into cDNA. Hexamers have a low melting temperature, which results in lower synthesis (40%) of cDNA from template RNA. Multivalent beads can be used to improve cDNA conversion. Fig.12 An example of an oligonucleotide N-mer (random oligonucleotide) for bulk cDNA synthesis is shown, while Fig.13 Specific oligonucleotide probes in targeted cDNA synthesis are shown. Fig.13 In the embodiment, the target nucleic acid 14 (here RNA) is bound to the multivalent assembly 10b having the oligonucleotide probe 20b specific to (complementary to) the target nucleic acid 14, while the other multivalent assemblies 10a, 10c without the probe specific to the target nucleic acid 14 remain unbound. The bound target 14 can be used to synthesize cDNA from the probe (e.g., probe 20b). The covalently attached cDNA also provides additional benefits, including physical separation, enabling the distinction of RNA / DNA co-assays, enrichment or depletion of target RNA, and buffer / enzyme exchange (single tube assay).

[0093] Fig.14 Shows the use of Fig.12 The reverse transcription workflow of cDNA synthesis of the multivalent assembly 10 shown.After using oligonucleotide 20 to capture RNA molecule 150 (for example, single-stranded RNA or messenger RNA), for example, using reverse transcriptase to extend complementary chain 154 from the 3' end of fixed oligonucleotide 20. Oligonucleotide 20 is shown as 15 aggressiveness, but other lengths can also be imagined, such as length is 6 to 30 bases, 10 to 20 bases, 15 to 25 bases. Therefore, the complementary cDNA chain of the extension of gained is coupled to pearl 5. In one embodiment, oligonucleotide 20 can include universal adapter sequence 160 as a part of joint sequence at 5' end. After complementary chain 154 is extended, chain 3' end can be adapted via adding 3' adapter (for example, via primer connection or amplification with complementary sequence of desired 3' adapter). Immobilized chain can be amplified as a part of library preparation step, and the amplified chain can be provided as input of NGS sequencing reaction.

[0094] Example

[0095] Fig.15Hybridization-based extraction was shown to outperform total nucleic acid extraction. The multivalent bead assembly was compared to a standard purification method using a commercially available kit in the COVID-Seq assay. The multivalent beads increased sensitivity and reduced the number of steps and touch points required. This was performed without bead optimization or oligonucleotide probe design, but still demonstrated a clear benefit in capturing low copy number nucleic acids from unpurified samples.

[0096] Figure 16 to Figure 17 show the experimental workflow and results of measuring the use of melting temperature as an agent of increased avidity. 400 base pairs (Cy5) single-stranded amplicon of internal labeling are used as targets, and each 20bp is designed to add an oligonucleotide probe of a joint (20Ts) to capture the target. The probe of design is coupled to a neutralizing avidin magnetic bead. The melting temperature of the oligonucleotide / target duplex is measured for the binding strength or avidity of the comparison multivalent beads. Fig.17A Example bead constructs are shown. Fig. 17B In the present invention, the melting temperatures of beads having 1 probe type per bead ("1 probe type beads"), 3 probe types per bead ("3 probe type beads"), and 5 probe types per bead ("5 probe type beads") are quantified. Probe type refers to a unique oligonucleotide sequence. Thus, 5 probe type beads include 5 different oligonucleotide probes with corresponding different sequences, while 1 probe type beads include a single probe type. However, where applicable, each bead may include multiple copies of each different probe type, or a single probe type. If the probes are not coordinated, the melting temperature of the single probe type beads should be the same as the melting temperature of the 5 probe type beads. If there is coordination, the melting temperature of the 1 probe type beads will be lower than that of the 5 probe type beads. This coordination is observed, where a single probe type has a lower melting temperature relative to 3 probe type beads and 5 probe type beads, showing an enhanced affinity with beads containing 3 oligonucleotide types and / or 5 oligonucleotide types.

[0097] Fig.18 Shown is an enrichment scheme suitable for bead enrichment. Lambda phage libraries were produced using the NexteraFlex library preparation scheme in the NexteraFlex enrichment kit, and then a modified enrichment scheme was used in the NexteraFlex enrichment kit. The modification was to add affinity beads coupled to the probe (as provided herein), rather than the probe in solution. Affinity beads were added after the initial 95°C and gradually reduced to 56°C. The final modification omitted the conventional capture step after enrichment.

[0098] Fig.19AA schematic diagram of a heteropolyvalent assembly with 5 different probes (e.g., 5 different probe sequences) attached to a single bead versus 5 different probe sequences separated on corresponding different beads versus 1 probe sequence on one bead is shown. A set of 5 probes of approximately 20 nucleotides in length with a 5' biotin poly A linker was designed. It should be understood that the 5 different probe types refer to available sequences and each bead can have multiple copies of the available sequences. Therefore, a bead with 5 different probe types can have multiple copies of each of the 5 different oligonucleotide sequences of the probe on the bead, while a bead with a single probe type can have multiple copies of a single probe type. The multivalent beads were compared to an 80-mer in solution that was aligned to the same region. Using Fig.18 The workflow mentioned in Fig.19B and Fig.19C As shown, higher on-target enrichment was observed for all five probes on a single bead compared to each probe separated on a different bead. Fig.19C The percentage of library fragments overlapping with the target region within 500bp is illustrated. The three columns on the left side show the high level enrichment of 5 probes fixed on the same beads, and the high level does not depend on the length of the joint, and three different joint lengths (20nt, 10nt, 0nt) are compared. The red column is the enrichment of 5 probes on 5 different beads, which has a lower enrichment efficiency. And the green bar shows a poor enrichment, with only 1 probe in 5 probes in the enrichment. The gray bar is a comparison with the 80nt probe. This provides evidence that the enhanced affinity improves the capture efficiency on the target.

[0099] Fig. 20A Multivalent beads are shown to improve read enrichment and enable hybridization to both strands. Using the lambda phage model, FWD or reverse (REV, reverse complement of FWD) were evaluated on beads alone or in solution relative to the continuous or non-multivalent case of an 80-mer forward (FWD) oligonucleotide probe alone. Enhancement of on-target enrichment relative to the 80-mer oligonucleotide probe was observed on beads, and inhibition was observed in solution. Fig. 20B is a schematic diagram of the experimental workflow.

[0100] The term "target or target nucleic acid" as disclosed herein can refer to a nucleic acid having a sequence of interest that hybridizes to an oligonucleotide of a multivalent assembly as provided herein. The term includes nucleic acid sequences whose subsequences bind to a multivalent assembly. In one embodiment, the target nucleic acid is a fragment generated as part of an NGS workflow.

[0101] Oligonucleotide disclosed herein is hybridized with the target nucleic acid in nucleic acid sample.Hybridization is carried out between single-stranded nucleic acid sequences.This can be realized by many methods well known in the art, such as using heat to denature or separate the complementary strand of double-stranded nucleic acid, which can hybridize with probe when cooling.In addition, when set 12 is provided as a part of probe group (for example, group 100), wherein each different target region is separated in space, probe group can be stored together and be provided in a single reaction vessel.Alternatively, the independent probe set of probe group can be stored separately.

[0102] As disclosed herein, oligonucleotides (e.g., probes) are nucleic acids that can bind to a target nucleic acid by one or more types of chemical bonds (usually by complementary base pairing, usually by hydrogen bond formation). Oligonucleotides can include natural or modified bases, and can be RNA or DNA. In addition, bases can be connected by bonds other than phosphodiester bonds, as long as they do not interfere with hybridization. Therefore, oligonucleotides can also be peptide nucleic acids (PNAs), in which the constituent bases are bound by peptide bonds rather than phosphodiester bonds.

[0103] The target nucleic acid may be derived from any in vivo or in vitro source, including from one or more cells, tissues, organs, or organisms (whether living or non-living) 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, which is derived from or is derived from humans, animals, plants, fungi (e.g., molds or yeasts), bacteria, viruses, viroids, mycoplasmas, 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 part of a chromosome, e.g., from one or more genes or loci of a chromosome), mitochondrial DNA, chloroplast DNA, plasmid or other episome-derived DNA (or recombinant DNA contained therein), or double-stranded cDNA, which is prepared by reverse transcribing RNA using an RNA-dependent DNA polymerase or reverse transcriptase to generate a 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 a plurality of dsDNA molecules in or prepared from a nucleic acid molecule (e.g., a plurality of dsDNA molecules in or prepared from a genomic DNA or cDNA prepared from RNA in or from a biological source (e.g., a cell, tissue, organ, organism) or an environmental source (e.g., water, air, soil, saliva, sputum, urine, feces). 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 prepared in vitro from single-stranded DNA (ssDNA) or from single-stranded or double-stranded RNA (e.g., using methods 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, which is prepared from all or part of one or more double-stranded or single-stranded DNA or RNA molecules using any method known in the art, including methods for the following operations: DNA or RNA amplification (e.g., PCR or reverse transcriptase-PCR (RT-PCR), transcription-mediated amplification methods, wherein all or part of one or more nucleic acid molecules are amplified); cloning all or part of one or more nucleic acid molecules into plasmids, fosmids, BACs or other vectors that are subsequently replicated in suitable host cells; or capturing one or more nucleic acid molecules by hybridization, such as by hybridization with DNA probes on an array or microarray. Target nucleic acids as provided herein may include, but are not limited to, DNA, RNA, peptide nucleic acids, morpholino nucleic acids, locked nucleic acids, glycol nucleic acids, threose nucleic acids, mixtures thereof, and hybrids thereof.In one embodiment, a genomic DNA fragment or a copy of its amplification is used as a target nucleic acid. In another embodiment, mitochondrial or chloroplast DNA is used. Other embodiments target RNA or its derivatives, such as mRNA or cDNA. In some embodiments, the target nucleic acid can be from a single cell. In some embodiments, the target nucleic acid can be from a cell-free body fluid, such as plasma or sputum lacking cells. In some embodiments, the target nucleic acid can be from circulating tumor cells.

[0104] The disclosed multivalent assemblies may include synthetic sequences or non-naturally occurring sequences that are not specific for any target sequence of the target source to reduce nonspecific binding.

[0105] This written description uses examples, including the best mode, and also enables any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems and performing any incorporated methods. 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 language of the claims.

Claims

1. A multivalent assembly, comprising: a first single-stranded oligonucleotide probe complementary to a first region of the target nucleic acid; a second single-stranded oligonucleotide probe complementary to a second region of the target nucleic acid; as well as A third single-stranded oligonucleotide probe complementary to a third region of the target nucleic acid, and wherein the melting temperatures of the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe from the target nucleic acid are all within 20 degrees Celsius. 2 . The multivalent assembly according to claim 1 , wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe have a length of 20 to 30 nucleotides. 3 . The multivalent assembly according to claim 1 , wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are immobilized on a surface via corresponding linkers. The multivalent assembly of claim 3 , wherein the corresponding linkers comprise universal adapter sequences. The multivalent assembly according to claim 3 , wherein the surface comprises a bead surface or a flat surface. 6 . The multivalent assembly according to claim 5 , wherein the bead surface comprises multiple copies of the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe. 7 . The multivalent assembly according to claim 1 , wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are coupled to a polypeptide.

8. The multivalent assembly according to claim 1, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are coupled to nucleic acid tiles. 9 . The multivalent assembly according to claim 1 , wherein the first region, the second region, and the third region of the target nucleic acid are continuous.

10. The multivalent assembly of claim 1, wherein the first region and the second region of the target nucleic acid are separated by 5 or fewer nucleotides. The multivalent assembly according to claim 10 , wherein the second region and the third region of the target nucleic acid are separated by 5 or fewer nucleotides.

12. The multivalent assembly according to claim 1, comprising a fourth single-stranded oligonucleotide probe, wherein the fourth single-stranded oligonucleotide probe comprises a fourth probe capture region complementary to a fourth region of the target nucleic acid, wherein the melting temperature of the fourth single-stranded oligonucleotide probe from the target nucleic acid is within 20 degrees Celsius.

13. The multivalent assembly according to claim 12, comprising a fifth single-stranded oligonucleotide probe, wherein the fifth single-stranded oligonucleotide probe comprises a fifth probe capture region complementary to a fifth region of the target nucleic acid, wherein the melting temperature of the fifth single-stranded oligonucleotide probe from the target nucleic acid is within 20 degrees Celsius. 14 . The multivalent assembly according to claim 1 , wherein the lengths of the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are different relative to each other.

15. A multivalent bead assembly, wherein the multivalent bead assembly include: Bead surface; a first single-stranded oligonucleotide probe comprising a first hybridization sequence complementary to a first region of a target nucleic acid; a second single-stranded oligonucleotide probe comprising a second hybridization sequence complementary to a second region of the target nucleic acid; and A third single-stranded oligonucleotide probe, the third single-stranded oligonucleotide probe comprising a third hybridization sequence complementary to a third region of the target nucleic acid, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe and the third single-stranded oligonucleotide probe are randomly immobilized on the bead surface, and wherein the first hybridization sequence, the second hybridization sequence and the third hybridization sequence are different from each other. 16 . The multivalent bead assembly according to claim 15 , wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are 20 to 30 nucleotides in length. 17 . The multivalent bead assembly according to claim 15 , wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are immobilized on the bead surface via corresponding linkers.

18. The multivalent bead assembly of claim 15, wherein the corresponding linker comprises a universal linker.

19. The multivalent bead assembly of claim 15, wherein the first region, the second region, and the third region of the target nucleic acid are continuous.

20. The multivalent bead assembly of claim 15, wherein the first region and the second region of the target nucleic acid are separated by 5 or fewer nucleotides.

21. The multivalent bead assembly of claim 15, wherein the second region and the third region of the target nucleic acid are separated by 5 or fewer nucleotides.

22. The multivalent bead assembly of claim 15, wherein oligonucleotide probes to different target nucleic acids are not immobilized on the bead surface.

23. The multivalent bead assembly of claim 15, wherein a subset of the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are arranged on the bead surface such that the target nucleic acid hybridizes to an individual first single-stranded oligonucleotide probe, an individual second single-stranded oligonucleotide probe, and an individual third single-stranded oligonucleotide probe.

24. The multivalent bead assembly of claim 15, comprising the target nucleic acid.

25. The multivalent bead assembly of claim 24, wherein the target nucleic acid is an RNA molecule.

26. The multivalent bead assembly of claim 15, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are random oligonucleotides. 27 . The multivalent bead assembly of claim 15 , wherein the length of the first single-stranded oligonucleotide probe is different from the length of one or both of the second single-stranded oligonucleotide probe and the third single-stranded oligonucleotide probe.

28. A hybridization kit, comprising: A first bead, the first bead comprising: a first plurality of single-stranded oligonucleotide probes randomly immobilized on the surface of the first bead, wherein a first subset of the first plurality of single-stranded oligonucleotide probes is complementary to a first region of a first target nucleic acid, a second subset of the first plurality of single-stranded oligonucleotide probes is complementary to a second region of the first target nucleic acid, and a third subset is complementary to a third region of the first target nucleic acid; and A second bead, the second bead comprising: a second plurality of single-stranded oligonucleotide probes randomly immobilized on the surface of the second bead, wherein a first subset of the second plurality of single-stranded oligonucleotide probes is complementary to a first region of a second target nucleic acid, a second subset of the second plurality of single-stranded oligonucleotide probes is complementary to a second region of the second target nucleic acid, and a third subset of the second plurality of single-stranded oligonucleotide probes is complementary to a third region of the second target nucleic acid.

29. The hybridization kit of claim 28, wherein the first bead and the second bead are part of a bead library.

30. The hybridization kit of claim 28, wherein a fourth subset of the first plurality of single-stranded oligonucleotide probes is complementary to a fourth region of the target nucleic acid, and wherein a fifth subset of the first plurality of single-stranded oligonucleotide probes is complementary to a fifth region of the target nucleic acid. The hybridization kit according to claim 28 , wherein the first target nucleic acid and the second target nucleic acid are complementary to each other.

32. A method for target enrichment, the method comprising: The nucleic acid fragments are contacted with a plurality of multivalent assemblies to form multivalent assembly-target nucleic acid complexes, wherein the multivalent assemblies include a set of individual probes specific for the corresponding target nucleic acids, the set of individual probes including: a first oligonucleotide probe complementary to a first subregion of the target nucleic acid; a second oligonucleotide probe complementary to a second subregion of the target nucleic acid; and a third oligonucleotide probe complementary to a third subregion of the target nucleic acid, wherein the first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe have sequences that are distinguishable from each other; and The multivalent assembly-target nucleic acid complex is separated from the non-hybridized nucleic acid fragments of the nucleic acid fragments to produce separated nucleic acid fragments.

33. The method of claim 32, comprising sequencing the purified nucleic acid fragments.

34. The method of claim 32, wherein prior to said contacting, adapters are added to the ends of said nucleic acid fragments.

35. The method of claim 32, wherein separate multivalent assemblies comprise separate sets of probes immobilized on the surface of a bead.

36. The method of claim 32, wherein the separating comprises magnetic separation to capture the beads.

37. The method of claim 32, wherein the separating comprises capturing an affinity binder of the multivalent assembly.

38. The method of claim 32, wherein separate multivalent assemblies comprise separate sets of probes immobilized on DNA tiles.

39. The method of claim 32, wherein separate multivalent assemblies comprise separate sets of probes immobilized on a polypeptide scaffold.

40. The method of claim 32, comprising producing the nucleic acid fragment.

41. A method for cDNA synthesis, the method comprising: The RNA sample is contacted with a multivalent assembly to capture RNA molecules, the multivalent assembly comprising a probe set immobilized on the surface of a bead, wherein the probe set comprises: a first oligonucleotide probe complementary to a first subregion of the RNA molecule; a second oligonucleotide probe complementary to a second subregion of the RNA molecule; and a third oligonucleotide probe complementary to a third subregion of the RNA molecule, wherein the first oligonucleotide probe, the second oligonucleotide probe and the third oligonucleotide probe have sequences that can be distinguished from each other; and The first oligonucleotide probe is extended using a reverse transcriptase to generate a cDNA complementary to at least a portion of the RNA molecule.

42. The method of claim 41, wherein the first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe are random N-mers of at least 15 bases in length.

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