Multivalent assemblies for enhanced target hybridization

Abstract

Multivalent assemblies for target hybridization are described. The multivalent assemblies include oligonucleotide sets that hybridize to target nucleic acids, allowing for capture of the target nucleic acids. In one embodiment, the multivalent assemblies are heteromultivalent, such that the oligonucleotide sets include different oligonucleotides that bind to different regions of the target nucleic acid.

Description

[Technical Field]

[0001] 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, it should not be assumed that the problems mentioned in this section or problems associated with the subject matter provided as background have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which, as such, may also correspond to embodiments of the claimed technology.

[0002] Sequencing methods on next-generation sequencing (NGS) platforms typically utilize nucleic acid fragment libraries. In targeted sequencing techniques, a subset of fragments containing a gene or region of interest in the genome is isolated from the nucleic acid library and sequenced. Targeted approaches using NGS allow researchers to focus time, cost, and data analysis on desired regions of interest. Such targeted analyses can include the exome (the protein-coding portion of the genome), specific genes of interest (custom content), intragenic targets, or mitochondrial DNA. Targeted approaches contrast with whole-genome sequencing approaches, which are more comprehensive but also include sequencing regions of the genome that may not be of interest to all users.

[0003] In one example of a target sequencing technique, target enrichment or hybridization pull-out uses a panel or set of probes that hybridize to target sequences in a nucleic acid library. Hybridization of the probes to the target sequences separates these sequences from other fragments in the library, and the captured sequences can be used to enhance target sequencing. Summary of the Invention

[0004] In one embodiment, the present disclosure provides a multivalent assembly, comprising 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 a range of 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 having a first hybridization sequence complementary to a first region of a target nucleic acid, a second single-stranded oligonucleotide probe having a second hybridization sequence complementary to a second region of the target nucleic acid, and a third single-stranded oligonucleotide probe having a third hybridization sequence complementary to a 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 immobilized 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 first beads comprising a first plurality of single-stranded oligonucleotide probes randomly immobilized on the surface of the first beads, wherein a first subset of the first plurality is complementary to a first region of a first target nucleic acid, a second subset of the first plurality 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 second beads comprising a second plurality of single-stranded oligonucleotide probes randomly immobilized on the surface of a second bead, wherein a first subset of the second plurality is complementary to a first region of a second target nucleic acid, a second subset of the second plurality is complementary to a second region of the second target nucleic acid, and a third subset of the second plurality 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 nucleic acids in a sample to generate nucleic acid fragments containing a target nucleic acid, and contacting the nucleic acid fragments with a plurality of multivalent assemblies to form multivalent assemblies-target nucleic acid complexes, each of which contains a respective set of probes specific to the target nucleic acid. Each of the probe sets includes 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 one another. The method also includes separating the multivalent assemblies-target nucleic acid complexes from unhybridized nucleic acid fragments of the nucleic acid fragments to generate 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 ensemble to capture RNA molecules, the multivalent ensemble comprising a probe set immobilized on the surface of beads. 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. The first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe have sequences that are distinguishable from one another. The method also includes extending the first oligonucleotide probe using a reverse transcriptase enzyme to generate cDNA complementary to at least a portion of the RNA molecule.

[0009] The foregoing description is presented to enable one to make and use the disclosed technology. Various modifications to the disclosed embodiments will be apparent, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosed technology. Thus, the disclosed technology is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. The scope of the disclosed technology is defined by the appended claims. [Brief explanation of the drawings]

[0010] These and other features, aspects, and advantages of the present invention will be better understood from the following detailed description when read in conjunction with the accompanying drawings, in which like features represent like parts throughout. [Figure 1] FIG. 1 shows the relationship between oligonucleotide length and full-length synthesis. [Figure 2] FIG. 1 shows the failed synthesis of an 80-mer oligonucleotide having a hairpin structure. [Figure 3] FIG. 1 is a schematic diagram of a multivalent assembly according to an aspect of the present disclosure. [Figure 4] FIG. 1 shows oligonucleotide probes of different lengths that fall within a melting temperature range. [Figure 5] FIG. 1 is a schematic diagram of an exemplary multivalent assembly having oligonucleotides immobilized on the surface of a bead, according to an aspect of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram of an exemplary multivalent assembly having oligonucleotides immobilized on beads, according to aspects of the present disclosure. [Figure 7] FIG. 1 is a schematic diagram of an exemplary surface of a multivalent assembly with randomly seeded oligonucleotides, according to an aspect of the present disclosure. [Figure 8] FIG. 1 is a schematic diagram of an exemplary multivalent assembly comprising a nucleic acid tile scaffold, according to aspects of the present disclosure. [Figure 9] FIG. 1 is a schematic diagram of an exemplary multivalent assembly comprising a polypeptide scaffold, according to aspects of the present disclosure. [Figure 10] FIG. 1 illustrates an exemplary target enrichment workflow using multivalent ensembles, according to aspects of the present disclosure. [Figure 11] FIG. 1 shows an exemplary cell-free nucleic acid capture using multivalent assemblies, according to aspects of the present disclosure. [Figure 12] FIG. 1 shows an exemplary nucleic acid capture using a multivalent assembly with random N-mers, according to an embodiment of the present disclosure. [Figure 13] FIG. 1 shows an exemplary nucleic acid capture using a multivalent assembly with target oligonucleotides, according to aspects of the present disclosure. [Figure 14] FIG. 1 shows an exemplary cDNA extension using multivalent assemblies, according to an embodiment of the present disclosure. [Figure 15] FIG. 1 shows that hybridization-based extraction outperforms total nucleic acid extraction. [Figure 16A] FIG. 1 shows the experimental workflow for determining the use of melting temperature as a surrogate for increased avidity. [Figure 16B] Similar to Figure 16A, images of the resulting decrease in binding as a function of temperature are shown for a fluorescently tagged target. [Figure 17A]FIG. 1 shows exemplary 1, 3, and 5 probe beads. [Figure 17B] FIG. 1 shows melting temperatures for 1, 3, and 5 probe beads showing binding modulation for multivalent assemblies with different probe types. [Figure 18] FIG. 1 shows a concentration protocol suitable for bead concentration. [Figure 19A] FIG. 1 shows exemplary hetero-multivalent and monovalent assemblies. [Figure 19B] FIG. 1 shows a comparison of target enrichment between hetero-multivalent and monovalent assemblies. [Figure 19C] FIG. 1 shows a comparison of target enrichment between hetero-multivalent and monovalent assemblies. [Figure 20A] FIG. 10 shows multivalent bead read enrichment for forward and reverse strands. [Figure 20B] FIG. 1 shows the lambda phage library workflow for multivalent beads versus in-solution probes. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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 particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosed technology. Thus, the disclosed technology is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0012] The use of nucleic acids with specificity for target sequences enables target enrichment, amplification, purification, extension, or other reactions. Capturing a subset of nucleic acid sequences with hybridization probes from biological samples or from libraries modified with sequencing adapters is used for enrichment in NGS and array-based profiling, as well as for applications such as pathogen detection and disease monitoring. Assays are designed using oligonucleotide probes or binders with high binding affinity and high specificity for the desired analyte compared to other oligonucleotide sequences. Enrichment techniques can employ a workflow that includes hybridization of a biotinylated probe to the nucleic acid sequence of interest, pull-down of the biotinylated probe onto streptavidin-functionalized magnetic beads, washing of the beads to remove nonspecifically bound molecules, elution of the target nucleic acid from the beads, and sequencing.

[0013] In these assays, relatively long nucleic acids can provide the desired target specificity compared to short nucleic acids and reduce the incidence of off-target binding. The binding strength, or avidity, of a nucleic acid to a target increases with the number of complementary nucleotides. However, longer nucleic acids used in specific binding reactions are relatively more expensive and complex to synthesize. Figure 1 shows the relationship between oligonucleotide length and the percentage of the produced product that represents the full-length oligonucleotide. As the length of the oligonucleotide increases, the production yield of the desired full-length product decreases. In certain cases, consecutive enrichment probes can be approximately 80-120 nucleotides. For high-throughput oligonucleotide synthesis, stepwise yields can range from 88-90%, with a final product yield of 15-30% for 80-120 base-long oligonucleotides with minimal secondary structure. As shown in Figure 2, T-type oligonucleotides, such as G-quadruplexes, are often used. m Oligonucleotides with higher secondary structure will result in poorer synthesis yields.

[0014] During manufacturing, only full-length probes are selected for use in the assay. Consequently, most of the synthesized product is lost, making the production of oligonucleotides with 80–120 nucleotides less efficient than shorter oligonucleotides. In applications using workflows with high levels of excess oligonucleotide probe relative to sample input, low probe production efficiency can pose additional challenges. In hybridization capture enrichment applications, a high library input of 200–500 ng (which may then include a preamplification step) and a high oligonucleotide probe concentration of 2000-fold molar excess can be used to achieve the desired target enrichment. Even with excess probe concentration, these longer probes experience variable enrichment due to secondary structures at elevated temperatures, leading to insufficient synthesis and capture of the target region. Therefore, generating sufficient probe concentration to satisfy high levels of excess probe in a particular workflow can be challenging, especially for probe lengths associated with low manufacturing yields.

[0015] Provided herein are multivalent assemblies, nucleic acids, reagents, kits, probe panels, and methods for producing and using multivalent assemblies. Multivalent assemblies can be part of an isolation reaction or can be used as part of a larger workflow (e.g., a sequencing workflow). The disclosed technology provides tailored short oligonucleotides that avoid low target affinity and improve synthesis yields compared to longer probes. Multivalent assemblies are provided that use multiple shorter probes targeting the same general region of a target nucleic acid compared to a single long probe. The technology provides cooperatively binding oligonucleotide sets (e.g., probe sets) that include these split or shorter oligonucleotides that bind to different regions of the target nucleic acid. In one embodiment, the target region of the target nucleic acid is composed of shorter subregions targeted by these separate oligonucleotides. This tailoring potentially increases hybridization strength (avidity), reduces probe and input requirements, increases wash stringency for nonspecific dissociation, and avoids potential secondary structures associated with longer probes that can lead to reduced production efficiency and poor target capture.

[0016] In embodiments, oligonucleotides can be used in conjunction with multivalent surfaces, heteromultivalent surfaces (e.g., beads), or branched oligonucleotides, each of which addresses the problem of inefficient probe-based capture of nucleic acids. Multivalent assemblies contain a set of unique oligonucleotide sequences, each capable of binding to a different stretch of a nucleic acid region. Multivalent probe structures (e.g., beads, branched oligonucleotides, or oligonucleotide handles) coordinate the binding of separate, unique oligonucleotide sequences, each capable of binding to a different stretch of the target. This coordinated hybridization improves the avidity of the multivalent structure for its target without the need to increase the length of each individual probe. Increased avidity also improves hybridization kinetics, allowing for faster annealing times compared to longer probes and reducing the required concentrations of probe and input in the hybridization reaction. In embodiments, the use of variable-length probes within multivalent assemblies avoids secondary structure and improves uniformity of hybridization intensity.

[0017] In embodiments, the use of a set of cooperatively binding oligonucleotides provides binding specificity to a target nucleic acid comparable to that of a single, continuous probe spanning the same region of the target nucleic acid. This avoids the manufacturing complexities associated with longer probes while maintaining avidity. Furthermore, the present technology can achieve more efficient probe capture by using physically separated forward and reverse complementary probes, for example, on separate beads, while still using an assembly that hybridizes to both strands of a nucleic acid duplex. The presence of both forward and reverse complementary probes in the reaction solution makes it more difficult to target both strands in solution, as inter-probe hybridization occurs, reducing hybridization capture of the target region. To avoid inter-probe hybridization between forward and reverse strand probes, certain techniques target only one of the two strands, making hybridization capture difficult in low-input, PCR-free libraries. The disclosed technology facilitates targeting both strands of a duplex while avoiding undesired inter-probe hybridization. The disclosed technology avoids the reduced efficiency of probe synthesis for long or problematic sequences by separating the probe sequence and the immobilized probe at specific locations. That is, by dividing the probe into smaller segments, synthesis complexity is avoided, yet similar hybridization efficiency can be achieved using cooperative binding. In embodiments, the disclosed technology provides improved on-target enrichment compared to sequential oligonucleotide hybridization capture approaches, as demonstrated using the lambda genome as a model system.

[0018] 3 is a schematic diagram of a multivalent assembly 10 in which a set 12 of oligonucleotides 20 (e.g., oligonucleotide probes) having distinct sequences and binding specificities for a target nucleic acid 14 are immobilized on a surface 34. The oligonucleotide set 12 includes individual single-stranded oligonucleotides 20 (e.g., single-stranded oligonucleotide probes), each having complementarity to a different region of the target nucleic acid 14. In one embodiment, the set 12 is an oligonucleotide probe set. As provided herein, oligonucleotides 20 may refer to oligonucleotide probes or probes for a target enrichment workflow.

[0019] As provided herein, multivalent collection 10 provides multiple hybridization or binding sites for a target, such as target nucleic acid 14. Thus, in one embodiment, multivalent refers to a structure having two or more hybridization or binding sites. Individual hybridization sites can include individual oligonucleotides 20 separated from one another. Thus, in the illustrated embodiment, the individual hybridization sites, designated A', B', C', D', and N', are not contiguous and are not located on the same oligonucleotide strand. That is, in one embodiment, each individual oligonucleotide 20 that includes a hybridization site complementary to a region of target nucleic acid 14 has a respective 5' end and a 3' end. Thus, as provided herein, distinct oligonucleotides 22, 24, 26, 28, and 30 include different individual hybridization sites A', B', C', D', and N'. As illustrated, collection 10 is heteromultivalent, such that oligonucleotides 20 have distinct or unique nucleotide sequences relative to other oligonucleotides 20 in set 12. However, other multivalent configurations are also contemplated. For example, one or more of the individual oligonucleotides 20 of set 12 may contain the same sequence as others of set 12.

[0020] Each set 12 includes multiple separate oligonucleotides 20, each complementary to a different region of target sequence 14. In the illustrated example, oligonucleotides 22, 24, 26, and 28 (having hybridization sites A', B', C', D', and N') represent different oligonucleotides 20 of set 12. Oligonucleotide 30 represents one or more additional oligonucleotides. Oligonucleotides 22, 24, 26, 28, and 30 are complementary to respective target binding regions 23, 25, 27, 29, and 31 (also designated regions A, B, C, D, and N) of target nucleic acid 14. The target binding regions represent different subregions of target nucleic acid 14. In one embodiment, target nucleic acid 14 can be a single-stranded nucleic acid or a nucleic acid fragment, and the target binding region as a whole encompasses only a portion of the fragment. That is, target nucleic acid 14 can include non-hybridizing regions that are not complementary to oligonucleotides 20. However, binding to oligonucleotide 20 allows for capture and enrichment of bound fragments including any unhybridized regions.

[0021] In one embodiment, each oligonucleotide 20 can be 10 to 80 nucleotides in length. For example, each individual oligonucleotide 20 in 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 in length. The complementary target-binding regions on target nucleic acid 14 are relatively shorter (e.g., 10 to 80 nucleotides in one embodiment) than the binding regions of conventional continuous or single nucleic acid hybridization probes, which can be 100 to 300 nucleotides in length. However, the span or length of all of target-binding regions 23, 25, 27, 29, and 31 as a whole 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 entire length of oligonucleotide 20 extending from the 5' end to the 3' end is complementary to its respective target-binding region. In other embodiments, one or more of the oligonucleotides 20 of set 12 may include a non-hybridizing region at the 5' and / or 3' end.

[0022] In one embodiment, oligonucleotides 20 are complementary to immediately adjacent or contiguous regions. For example, target binding regions 23, 25, 27, 29, and 31 can form a contiguous span or stretch of target nucleic acid 14. In other embodiments, a spacer region can be present between one or more of target binding regions 23, 25, 27, 29, and 31. By way of example, the spacer region can be 1 to 10 nucleotides long (e.g., 1 to 4 nucleotides, 1 to 5 nucleotides). In other examples, the spacer region can be longer to accommodate different multivalent assembly configurations. For example, a polypeptide base or DNA tile surface 34 can generate oligonucleotides that bind to target binding regions where at least two of the target binding regions are separated by 10 to 25 nucleotides or more. In one embodiment, target binding regions 23, 25, 27, 29, and 31 are non-overlapping and arranged in 5' to 3' order or vice versa.

[0023] In the illustrated example, oligonucleotide set 12 includes at least four distinct oligonucleotides 20. However, as generally described herein, it should be understood that set 12 may include a greater or lesser number. The number of unique oligonucleotides 20 in set 12 may depend on variables including the size and shape of surface 34 and the length of the optional spacer region. For example, in one embodiment, oligonucleotide set 12 includes a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide.

[0024] It should be understood that, in embodiments, multivalent collection 10 may be provided with oligonucleotides 20 in a single-stranded state, e.g., an unhybridized or unbound state. However, when a target nucleic acid 14 having a sequence complementary to some or all of the oligonucleotides 20 of set 12 is present under favorable hybridization conditions (e.g., temperature), the complementary oligonucleotides 20 may hybridize to target nucleic acid 14. Thus, when hybridized to target nucleic acid 14, oligonucleotides 20 are in a duplex or double-stranded state.

[0025] Surface 34 may be a bead 50 (see FIG. 5), such as a magnetic bead. In embodiments, surface 34 may be a substrate, a flow cell surface, a planar surface, a molded surface, a multi-well surface, a patterned surface, or a molecule, such as a DNA tile structure (see FIG. 8) or a polypeptide (see FIG. 9).

[0026] Target binding to set 12 involves multiple shorter binding interactions to corresponding shorter target binding regions, meaning that multivalent ensemble 10 has binding kinetics that more closely resemble those of shorter nucleotides. In one embodiment, multivalent ensemble 10 can achieve target specificity comparable to or approaching that of conventional probes with improved binding kinetics of shorter nucleic acids.

[0027] As provided herein, an oligonucleotide set 12 or multivalent collection 10 may include two or more oligonucleotides 20, whereby each individual oligonucleotide 20 of a set 12 is complementary to a respective portion of a target nucleic acid 14, e.g., a single-stranded target nucleic acid 14. However, within a particular set 12, the individual oligonucleotides 20 may have non-uniform or different lengths (e.g., different nucleotide lengths). The length of each of the oligonucleotides 20 can be selected to achieve a desired melting temperature (Tm) or to be within a particular melting temperature range.

[0028] FIG. 4 shows an exemplary group of oligonucleotides of varying lengths, yet having melting temperatures within a particular Tm range, such that the difference between the lowest and highest Tm of set 12 is no greater than a predetermined value. In one embodiment, the melting temperatures of set 12 are within 20° C. or 10° C. of each other. In one embodiment, the oligonucleotides of set 12 are of varying lengths, but may be within a predetermined length range and have melting temperatures within a particular Tm range. In one embodiment, the longest oligonucleotide 20 of each set 12 may be 5-15 nucleotides or 5-10 nucleotides longer than the shortest oligonucleotide 20 of each set 12. In one embodiment, the oligonucleotides 20 of each set 12 may be 20-30 nucleotides long. In one embodiment, at least one oligonucleotide 20 of each set 12 has a different length relative to the other oligonucleotides 20 of set 12. In one embodiment, all of the oligonucleotides 20 of each set 12 have a different length compared to the other oligonucleotides 20 of set 12.

[0029] In one embodiment, the oligonucleotides 20 in each set 12 have similar Tms at which they melt or dissociate from the target nucleic acid 14. Furthermore, in embodiments in which a panel of multiple different probe sets 12 is used (see FIG. 10), all of the different sets 12 can be designed so that all or most of the oligonucleotides 20 in each different probe set 12 fall within a predetermined estimated Tm range. In one embodiment, the estimated Tm range of the oligonucleotides is selected to be 50-70°C or 55-65°C. In one embodiment, the estimated Tm range of the oligonucleotides is selected so that all nucleotides in the set 12 have melting temperatures within a temperature range of 20°C of each other. In one embodiment, the estimated Tm range of the oligonucleotides is selected so that all nucleotides in the set 12 have melting temperatures within a temperature range of 1 ... are within 5-20°C of each other, 10-20°C of each other, or 15-20°C of each other. Probes of different or more variable lengths can result in more uniform Tm between probes. Additionally, certain multivalent assemblies 10 can include modified nucleic acids (e.g., locked nucleic acids) to improve stability and hybridization to target nucleic acids 14.

[0030] In one embodiment, the Tm of a particular individual oligonucleotide 20 of a probe set 12, or of a fully assembled multivalent assembly 10, can be estimated based on the following hypothetical nearest neighbor formula:

[0031]

number

[0032] [Table 1] Table 1: * The left sequence is 5' to 3' and the right sequence is 3' to 5'. For example, in the case of AA / TT, AA is 5' to 3' and TT is 3' to 5'. When selecting a value, always select 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 favored. Positive values ​​reflect the reverse reaction, i.e., melting, and the same T m This leads to calculations.

[0033] In another example, Tm can be estimated as follows: For sequences of less than 14 nucleotides, the formula is: Tm = (wA + xT) * 2+(yG+zC) * 4 In the formula, w, x, y, and z are the numbers of bases A, T, G, and C in the sequence, respectively. For sequences longer than 13 nucleotides, the formula used is: Tm=64.9+41 * (yG+zC-16.4) / (wA+xT+yG+zC) The above formula is for annealing with 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.

[0034] FIG. 5 shows an exemplary multivalent assembly 10 in which a set 12 of oligonucleotides 20 is immobilized on the surface 34 of a bead 50. The illustrated example is a heteromultivalent configuration in which multiple oligonucleotides with different sequences are immobilized on a single particle (e.g., a magnetic bead). The number of unique probes per particle can be determined empirically and depends on variables including the size of the particle and the length of the optional linker 36, as shown in FIG. 6. The linker 36 provides flexibility and length away from the bead 50, allowing probes to be arranged in the correct order to coordinate hybridization. The linker 36 can be a universal linker 36 that is the same for different oligonucleotides. The linker 36 can be a nucleotide linker, a chemical linker, or a polymer linker. In one embodiment, the disclosed multivalent assembly can include a hybridization probe set that can be used, for example, for target enrichment NGS. For hybridization capture, many particle types or multivalent assemblies (each with a unique probe set 12) are pooled together to enrich for a population of desired target regions.

[0035] As shown in Figure 7, the surface 34 of a bead or other structure can be randomly seeded with oligonucleotides 20 so that subsets 56 of oligonucleotides 20 are arranged in the correct order relative to one another to promote hybridization to target nucleic acids 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. Thus, each bead 50 or other collection 10 may be capable of hybridizing multiple targets, if available in a given sample. Each set 12 of oligonucleotides 20 can, in one embodiment, include multiple copies of each individual oligonucleotide 20 representing different heterovalent binding sequences.

[0036] In Figure 8, an exemplary multivalent assembly 10 includes oligonucleotides 20 immobilized on prefolded DNA tiles 60. DNA origami techniques are used to assemble tile bases with variable hook arms 64 of different nucleotide sequences available for hybridizing to linker sequences 66 attached to the oligonucleotides 20. This allows a single universal tile base 60 to be used with many different sets 12 to capture a variety of different targets in a pooled manner. Furthermore, the hooks 64 can be arranged so that the oligonucleotides 20 are sequentially arranged to hybridize with targets according to their sequence. For hybridization capture, many tiles (each with a unique probe set 12) are pooled together to enrich for a group of desired target regions. The tiles 60 are bound to affinity molecules 68, such as biotin, which bind to affinity molecule binders such as streptavidin, facilitating separation of the bound target nucleic acids 14 from other components of the sample.

[0037] Figure 9 shows an exemplary multivalent assembly including a polypeptide scaffold 70. Oligonucleotides 20 can be attached to a universal peptide (e.g., polypeptide scaffold 70) via multiple orthogonal chemical reactions in an ordered fashion. Peptide synthesis of alpha helices with several orthogonal amino acid chemical handles can be used to sequentially attach oligonucleotides 20. The oligonucleotides 20 can be ordered according to the target sequence to enable target capture. Binding to affinity molecules 68 allows for separation after hybridization. Several peptide:oligonucleotide multivalent assemblies 10, each with specificity for a different target nucleic acid 14 (e.g., with different sequences), can be pooled for parallel enrichment on desired target regions.

[0038] Multivalent assemblies 10 can include sets 12 of oligonucleotides 20 covalently attached at multiple branch points to a biotinylated scaffold, such as a dendrimer or bottlebrush. These multivalent assemblies 10 can hybridize to their targets in solution and be captured on streptavidin beads. In the bottlebrush approach, terminal deoxynucleotidyl transferase extends azide-linked nucleotides from ssDNA oligonucleotides, or DNA polymerase adds azide-linked nucleotides via template amplification. After incorporation of the azide-linked nucleotides, oligonucleotides 20 are covalently attached via azide-alkyne cycloaddition (click chemistry). The branched configuration, coupled to a more flexible scaffold compared to beads, improves coordination of the probes with their respective targets and also avoids precipitation or aggregation.

[0039] Figure 10 illustrates an exemplary target enrichment workflow using the multivalent assembly 10 provided herein. The terms enrichment or target enrichment refer to a process that increases the relative abundance of a specific nucleic acid sequence in a sample compared to the overall level of nucleic acid sequences initially present in the sample before processing. Thus, the enrichment step provides a percentage or partial increase rather than a direct increase in the copy number of the nucleic acid sequence of interest, as occurs with amplification methods such as PCR. The methods described herein can be used to remove DNA strands that are not desired for sequencing, rather than specifically amplifying only the sequence of interest. At the whole genome level, removing 50% of the DNA sample reduces the cost and time of sequencing the remaining regions of biological interest from the whole genome by half. The methods described herein can also be used to select large regions (e.g., megabases) of the genome for resequencing of multiple individuals, or to select all exons in a genome sample. Synthesis of a single array or pool of oligonucleotides can be used to process multiple samples of interest; therefore, the cost of oligonucleotide synthesis can be amortized over many individual samples.

[0040] The illustrated target enrichment workflow uses a panel 100 of multivalent collections 10, each specific for a different target nucleic acid 14. The panel 100 may include multivalent collections 10 capable of hybridizing to a selected group of different target sequences. 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 multivalent collection includes a set 12 of unique oligonucleotides 20 having sequences specific for a particular target sequence 14. Thus, all hybridization sequences of individual oligonucleotides 20 of the set 12 may be unique within the panel 100. In the illustrated example, the oligonucleotides 20 may function as hybridization probes.

[0041] As shown, the target nucleic acid 14 may be in the form of nucleic acid fragments 102. The nucleic acid fragments 102 provided herein, such as for target enrichment or amplification reactions, may include relatively large sequence fragments, such as 10 kilobases (kb) to 62 megabases (Mb) in length. In other embodiments, the fragments are less than about 1 kb in length, e.g., in the range of 100 to 1000 bases in length, or in the range of 450 to 750 bases in length. It will be apparent to one of skill in the art that the following non-limiting fragmentation methods may be used: tagmentation with restriction endonucleases, other suitable enzymes, transposases, mechanical forms of fragmentation, such as nebulization or sonication, or non-enzymatic chemical fragmentation.

[0042] The panel 100 and fragments 102 are contacted with each other in a hybridization step 112 under conditions that allow hybridization of the oligonucleotides 20 of the multivalent collection 10 to their respective target nucleic acids 14. Hybridization results in the formation of multivalent collection-target nucleic acid complexes 120 for at least some of the fragments 102 and at least some of the multivalent collections 10. That is, hybridization occurs when target nucleic acids 14 are present within the fragments 102. Furthermore, some of the fragments 102 may not contain any sequences targeted by the panel 100. In one embodiment, hybridization to target nucleic acids 14 provided herein (e.g., combining and / or assembling sets 12) is performed at 50°C to 65°C, with hybridization times of 3 hours or less, 2 hours or less, or 1 hour or less to achieve the desired level of target nucleic acid binding and avoid nonspecific binding.

[0043] The hybridization step 112 may include a denaturation step in which the fragments 102 and probe panel 100 are heated to at least 90°C (e.g., 90°C-95°C) to denature the fragments 102 and dissociate the nucleic acids of the multivalent assembly 10. The workflow may include a gradual or stepwise reduction in temperature to the desired hybridization temperature range. In one example, after denaturation, the temperature is reduced to below the melting temperature of the individual oligonucleotides (e.g., 50°C-65°C). This relatively low temperature allows binding of the individual oligonucleotides 20 of the set 12. 50°C-65°C is held for a relatively short time (e.g., 10-20 minutes). The temperature is then slowly increased to allow non-specifically bound probes to melt. Again, this relatively high temperature is held for a predetermined time (e.g., 10-20 minutes).

[0044] The hybridization step 112 can also be performed either on a solid surface 34 (e.g., on beads 50) or in solution. In certain embodiments, at least one nucleic acid of the multivalent assemblies 10 can have a modification or affinity binder 68 that facilitates separation of the bound fragments 102 from the unbound fragments 102. Thus, the multivalent assemblies 10 provided herein can be bound to affinity binding molecules 68 of binding pairs such as biotin / streptavidin, biotin / avidin, biotin / neutravidin, DNP / anti-DNP, DIG / anti-DIG, etc., with a specific antibody that binds to digoxigenin being an example of a specific binding pair. In one example, biotinylation of the nucleic acids of the multivalent assemblies 10 facilitates selection with 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, modifications are provided at the 5' or 3' end of the individual nucleic acids of the multivalent assemblies 10. The nucleic acids of multivalent assemblies 10 may also include a unique barcode or sequence (e.g., a unique molecular identifier) ​​to facilitate identification. In embodiments in which multivalent assemblies 10 include beads 50, beads 50 may be magnetic beads that can be separated using magnetic pulldown. In other embodiments, beads 50 may include affinity binders 68.

[0045] In certain embodiments, the hybridization step 112 can be performed in solution, and subsequent addition of beads bearing a matching affinity binder results in binding of the multivalent assemblies 10 carrying the affinity binder, either as duplexes with the fragments or as single strands. For example, this may be the case when the multivalent assemblies 10 comprise a DNA or polypeptide scaffold bound to an affinity binder. The multivalent assemblies 10 can hybridize to fragments 102 containing target sequences in solution, and the multivalent assemblies 10 can be captured via the affinity binder. Uncaptured fragments 102 can be removed from the beads, for example, by washing.

[0046] In one embodiment, the captured fragments can be removed from the probe-target complexes, for example, by elution, prior to sequencing. Removal of the selected targets from the immobilized capture probes by denaturation generally results in a solution of enriched target nucleic acid fragments 130. The enriched target nucleic acid fragments 130 can be provided for a subsequent sequencing step. In an alternative embodiment, the enriched target nucleic acid fragments 130 can be amplified while bound to the beads, for example, by emulsion phase PCR, or can be eluted from the beads and amplified in solution before binding to a surface as part of a sequencing reaction.

[0047] In one embodiment, fragments 102 may be fragments generated through a library preparation workflow and include terminal adapters 140 suitable for use in sequencing, e.g., enriched target nucleic acid fragments 130 may be captured on a solid support and may function as primer binding sites. Adapters 140 may be universal adapters, e.g., comprising a common sequence. In one embodiment, adapters 140 may be Illumina sequencing adapters (Illumina, Inc.). Thus, the common sequences of adapters 140 may tend to hybridize to each other. In such an embodiment, adapter blockers may also be used during hybridization step 112. Fragments may be obtained from a single library (i.e., singleplex) or multiplexed from multiple libraries.

[0048] In another embodiment, adapters 140 can be ligated to the enriched target nucleic acid fragments 130 after elution and further preparation steps. For example, the enriched target nucleic acid fragments 130 can also be further fragmented after elution from the beads 120 or other support. In one embodiment, it may be advantageous to capture relatively large fragments 102, e.g., fragments 102 with an average size of 10 kB, thereby requiring fewer probe sets 12 to select specific megabase regions. While 10 kB regions can be selected, they are not easily amplified, so they can be further fragmented to an average of several hundred bases after the enrichment step. If a second fragmentation step is used, universal adapters 140 can be ligated onto the enriched target nucleic acid fragments 130 after elution and further fragmentation steps.

[0049] In another embodiment, the disclosed multivalent assemblies 10 can be used for nucleic acid capture. Figure 11 shows an example of the capture of cell-free DNA and / or RNA from a biological sample using a bead-based multivalent assemblies 10. Using multivalent beads or other multivalent assemblies 10 to capture nucleic acids from a biological sample offers advantages over traditional purification approaches, such as column-based purification. The multivalent assemblies 10 couple the purification and enrichment of target fragments. In another example, probes can be used to deplete unwanted nucleic acids, such as abundant RNAs like ribosomal RNA, from a sample. Due to their high avidity, the multivalent assemblies 10 can capture target fragments with higher sensitivity at lower copy numbers compared to traditional approaches. Once nucleic acids are captured on the multivalent assemblies 10, they can be enzymatically manipulated to add sequences, such as sequencing adapters, to the bead surface. This allows for enzyme and buffer exchange while the nucleic acids are bound to the beads.

[0050] Multivalent assemblies 10 can be used in on-bead conversion of RNA to cDNA (i.e., cDNA conversion). Figures 12-13 illustrate nucleic acid capture by multivalent beads for converting RNA to a cDNA library. In conventional cDNA synthesis, hexamers are used to convert bulk RNA to cDNA. Hexamers have a low melting temperature, resulting in a low cDNA synthesis rate (40%) from template RNA. Multivalent beads can be used to improve cDNA conversion. Figure 12 shows an example of oligonucleotide N-mers (randomers) for bulk cDNA synthesis, and Figure 13 shows specific oligonucleotide probes for targeted cDNA synthesis. In Figure 13, a target nucleic acid 14 (here, RNA) binds to multivalent assemblies 10b, which have oligonucleotide probes 20b specific to the target nucleic acid 14 (complementary to the target nucleic acid 14), while other multivalent assemblies 10a and 10c, which do not have probes specific to the target nucleic acid 14, remain unbound. The bound target 14 can be used for cDNA synthesis from a probe (e.g., probe 20b). Covalently bound cDNA also offers additional advantages, including physical separation, allowing compartmentalization for simultaneous RNA / DNA assays, concentration or depletion of target RNA, and buffer / enzyme exchange (single-tube assays).

[0051] FIG. 14 illustrates a reverse transcription workflow for cDNA synthesis using the multivalent assembly 10 shown in FIG. 12. After capture of an RNA molecule 150 (e.g., single-stranded RNA or messenger RNA) using an oligonucleotide 20, a complementary strand 154 is extended from the 3' end of the immobilized oligonucleotide 20 using, for example, a reverse transcriptase. While the oligonucleotide 20 is shown as a 15-mer, other lengths, such as 6-30 bases, 10-20 bases, or 15-25 bases, are also contemplated. The extended complementary cDNA strand is then bound to a bead 5. In one embodiment, the oligonucleotide 20 can include a universal adapter sequence 160 at its 5' end as part of a linker sequence. Following extension of the complementary strand 154, the 3' end of the strand can be adapted via the addition of a 3' adapter (e.g., via ligation or amplification with a primer bearing the complement of the desired 3' adapter). The immobilized strands can be amplified as part of the library preparation process, and the amplified strands can provide input to an NGS sequencing reaction. [Example]

[0052] Figure 15 shows that hybridization-based extraction outperforms total nucleic acid extraction. Multivalent bead assembly was compared to standard purification methods using a commercially available kit in the COVID-Seq assay. Multivalent beads improved sensitivity and reduced the number of required steps and touchpoints. This was performed without optimizing bead or oligonucleotide probe design, yet demonstrated clear advantages in capturing low-copy-number nucleic acids from unpurified samples.

[0053] Figures 16-17 show the experimental workflow and results for measuring the use of melting temperature as a proxy for increased avidity. Using a 400-base-pair, internally labeled (Cy5) single-stranded amplicon as the target, 20-bp oligonucleotide probes and linkers (20Ts) were designed to capture the target. The designed probes were conjugated to NeutrAvidin magnetic beads. The binding strength or avidity of multivalent beads was compared by measuring the melting temperature of the oligonucleotide / target duplex. Figure 17A shows an exemplary bead construct. Figure 17B quantifies the melting temperatures of beads with one probe type per bead ("1-probe beads"), three probe types per bead ("3-probe beads"), and five probe types per bead ("5-probe beads"). Probe type refers to a unique oligonucleotide sequence. Thus, 5-probe beads contain five different oligonucleotide probes, each with a different sequence, while 1-probe beads contain a single probe type. However, each bead may contain multiple copies of each different probe type, or a single probe type, if applicable. If the probes are not coordinated and bound, the melting temperature of a single-probe bead should be the same as that of a five-probe bead. If coordination is present, the melting temperature of a single-probe bead will be lower than that of a five-probe bead. This coordination has been observed, and the single-probe type has a lower melting temperature compared to the three-probe and five-probe beads, indicating improved avidity with beads containing three and / or five oligonucleotide types.

[0054] Figure 18 shows an enrichment protocol suitable for bead enrichment. A lambda phage library was generated using the NexteraFlex library preparation protocol of the NexteraFlex enrichment kit, followed by a modification of the enrichment protocol of the NexteraFlex enrichment kit. The modification was the addition of avidity beads conjugated to probes (as described herein) instead of probes in solution. After the initial 95°C addition of the avidity beads, the temperature was reduced to 56°C. The final modification was the skipping of the traditional capture step after enrichment.

[0055] Figure 19A shows a schematic diagram of hetero-multivalent assembly, with five different probes (e.g., five different probe sequences) attached to a single bead, each separated on a different bead, and one probe sequence per bead. A set of five probes, approximately 20 nucleotides in length, with a 5' biotin polyA linker was designed. It should be understood that the five different probe types refer to available sequences, and each bead may have multiple copies of an available sequence. Thus, a bead with five different probe types may have multiple copies of each of the five different oligonucleotide sequences of the probes on the bead, while a bead with a single probe type may have multiple copies of a single probe type. Multivalent beads were compared with a solution 80-mer aligned to the same region. Using the workflow described in Figure 18, higher on-target enrichment was observed with all five probes on a single bead compared to each probe separated on a different bead, as shown in Figures 19B and 19C. Figure 19C is a graph showing the percentage of library fragments overlapping the target region within 500 bp. The three columns on the left show high levels of enrichment with five probes immobilized on the same beads, independent of linker length; three different linker lengths (20 nt, 10 nt, and 0 nt) were compared. The red columns show enrichment from five probes on five different beads, with lower enrichment efficiency. The green bars show worse enrichment with only one of the five probes in the enrichment. The gray bars show a comparison with the 80 nt probe. This provides evidence that increased avidity improves on-target capture efficiency.

[0056] Figure 20A shows that multivalent beads improve read enrichment and allow hybridization to both strands. Forward (FWD) or reverse (REV, reverse complement of FWD) orientations on separate beads or in solution were evaluated compared to a contiguous or non-multivalent 80-mer FWD-only oligonucleotide probe using a lambda phage model. Compared to the 80-mer oligonucleotide probe, improved on-target enrichment was observed on the beads, while suppression was observed in solution. Figure 20B is a schematic diagram of the experimental workflow.

[0057] The term target or target nucleic acid as disclosed herein may refer to a nucleic acid having a sequence of interest that hybridizes to an oligonucleotide of a multivalent collection provided herein. This term includes nucleic acid sequences whose subsequences bind to the multivalent collection. In one embodiment, the target nucleic acid is a fragment generated as part of an NGS workflow.

[0058] The oligonucleotides disclosed herein hybridize to target nucleic acids in a nucleic acid sample. Hybridization occurs between single-stranded nucleic acid sequences. This can be achieved by a number of methods well known in the art, such as using heat to denature or separate complementary strands of double-stranded nucleic acids, allowing them to hybridize to the probes upon cooling. Furthermore, when set 12 is provided as part of a probe panel (e.g., panel 100) in which each distinct target region is spatially separated, the probe panel can be stored together and provided in a single reaction vessel. Alternatively, individual probe sets of the probe panel can be stored separately.

[0059] The oligonucleotides disclosed herein, such as probes, are nucleic acids that can bind to target nucleic acids through one or more chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Oligonucleotides may contain natural or modified bases and may include RNA or DNA. Furthermore, bases may be linked by linkages other than phosphodiester bonds, as long as they do not interfere with hybridization. Thus, oligonucleotides may be peptide nucleic acids (PNAs) in which the constituent bases are linked by peptide bonds instead of phosphodiester bonds.

[0060] A "target nucleic acid" can be derived from any in vivo or in vitro source, including one or more 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 originating or 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 portion of a chromosome, e.g., from one or more genes or loci from a chromosome), mitochondrial DNA, chloroplast DNA, DNA from (or recombinant DNA contained within) a plasmid or other episome, or double-stranded cDNA made by reverse transcribing 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 a plurality of dsDNA molecules in or prepared from a nucleic acid molecule (e.g., a plurality of dsDNA molecules in or prepared from genomic DNA or cDNA prepared from RNA in or derived 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 prepared in vitro from single-stranded DNA (ssDNA) or from single- or double-stranded RNA (e.g., using methods 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 prepared from all or a portion of one or more double-stranded or single-stranded DNA or RNA molecules using any method known in the art, including amplification of DNA or RNA (e.g., PCR or reverse transcriptase PCR (RT-PCR), transcription-mediated amplification methods, involving 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 is then replicated in a suitable host cell; or methods for capture of one or more nucleic acid molecules by hybridization, such as hybridization to DNA probes on an array or microarray. Target nucleic acids 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 an amplified copy thereof is used as the target nucleic acid. In another embodiment, mitochondrial DNA or chloroplast DNA is used. Still other embodiments target RNA or its derivatives, such as mRNA or cDNA. In some embodiments, the target nucleic acid may be derived from a single cell. In some embodiments, the target nucleic acid may be derived from acellular body fluids, such as plasma or sputum, which are devoid of cells. In some embodiments, the target nucleic acid may be derived from circulating tumor cells.

[0061] The disclosed multivalent assemblies can include synthetic or non-naturally occurring sequences that are not specific for any target sequence of the target source, to reduce non-specific binding.

[0062] 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 device or system and performing any incorporated methods. Such other examples are intended to be within the scope of the claims if they include structural elements that do not differ from the literal words of the claims, or if they include equivalent structural elements that have insignificant differences from the literal words of the claims.

Claims

1. 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; a third single-stranded oligonucleotide probe complementary to a third region of the target nucleic acid, 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 a range of 20°C. Multivalent aggregates.

2. 2. The multivalent collection of claim 1, 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.

3. 2. The multivalent assembly of 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 their respective linkers.

4. The multivalent assembly of claim 3 , wherein each of the linkers comprises a universal adaptor sequence.

5. The multivalent assembly of claim 3 , wherein the surface comprises a bead surface or a planar surface.

6. 6. The multivalent assembly of 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. 2. The multivalent assembly of claim 1, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are bound to a polypeptide.

8. 2. The multivalent collection of claim 1, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are bound to a nucleic acid tile.

9. The multivalent assembly of claim 1 , wherein the first region, the second region, and the third region of the target nucleic acid are contiguous.

10. 2. The multivalent collection of claim 1, wherein the first region and the second region of the target nucleic acid are separated by no more than five nucleotides.

11. 11. The multivalent collection of claim 10, wherein the second region and the third region of the target nucleic acid are separated by no more than five nucleotides.

12. 2. The multivalent assembly of claim 1, comprising a fourth single-stranded oligonucleotide probe comprising 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 a range of 20°C.

13. 13. The multivalent assembly of claim 12, comprising a fifth single-stranded oligonucleotide probe comprising 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 a range of 20°C.

14. 2. The multivalent collection of claim 1, wherein the first single-stranded oligonucleotide probe, the second single-stranded oligonucleotide probe, and the third single-stranded oligonucleotide probe are of different lengths relative to each other.

15. a bead surface; a first single-stranded oligonucleotide probe comprising a first hybridization sequence complementary to a first region of the target nucleic acid; a second single-stranded oligonucleotide probe comprising a second hybridization sequence complementary to a second region of the target nucleic acid; a 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 surface of the bead, and the first hybridization sequence, the second hybridization sequence, and the third hybridization sequence are different from one another. Multivalent bead aggregates.

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 described in 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 their respective linkers.

18. 16. The multivalent bead collection of claim 15, wherein each of the linkers comprises a universal linker.

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

20. 16. The multivalent bead collection of claim 15, wherein the first region and the second region of the target nucleic acid are separated by no more than five nucleotides.

21. 16. The multivalent bead collection of claim 15, wherein the second region and the third region of the target nucleic acid are separated by no more than five nucleotides.

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

23. 16. The multivalent bead collection 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 each of the first single-stranded oligonucleotide probe, each of the second single-stranded oligonucleotide probe, and each of the third single-stranded oligonucleotide probe.

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

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

26. 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 randomers.

27. The multivalent bead collection of claim 15, wherein the first single-stranded oligonucleotide probe has a different length from one or both of the second single-stranded oligonucleotide probe and the third single-stranded oligonucleotide probe.

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

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

30. 29. The hybridization kit of claim 28, wherein a fourth subset of the first plurality is complementary to a fourth region of the target nucleic acid and a fifth subset of the first plurality is complementary to a fifth region of the target nucleic acid.

31. 30. The hybridization kit of claim 28, wherein the first target nucleic acid and the second target nucleic acid are complementary to each other.

32. 1. A method of target enrichment comprising: contacting the nucleic acid fragments with a plurality of multivalent assemblies to form multivalent assemblies-target nucleic acid complexes, the multivalent assemblies comprising individual probe sets specific for each target nucleic acid, each probe set comprising: 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 forming a third oligonucleotide probe complementary to a third subregion of the target nucleic acid, the first oligonucleotide probe, the second oligonucleotide probe, and the third oligonucleotide probe having sequences distinguishable from one another; and separating said multivalent assembly-target nucleic acid complexes from unhybridized nucleic acid fragments of said nucleic acid fragments to produce separated nucleic acid fragments.

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

34. 33. The method of claim 32, further comprising adding adaptors to the ends of the nucleic acid fragments prior to said contacting.

35. 33. The method of claim 32, wherein each multivalent ensemble comprises each of said probe sets immobilized on the surface of a bead.

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

37. 33. The method of claim 32, wherein said separating comprises capturing affinity binders of said multivalent ensemble.

38. 33. The method of claim 32, wherein each multivalent ensemble comprises each of said probe sets immobilized on a DNA tile.

39. 33. The method of claim 32, wherein each multivalent assembly comprises each of said probe sets immobilized on a polypeptide scaffold.

40. 33. The method of claim 32, comprising generating the nucleic acid fragments.

41. 1. A method for cDNA synthesis comprising: contacting an RNA sample with a multivalent ensemble to capture RNA molecules, the multivalent ensemble comprising a probe set immobilized on a surface of a bead, the probe set comprising: a first oligonucleotide probe complementary to a first subregion of said 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 are distinguishable from one another; extending said first oligonucleotide probe using a reverse transcriptase to generate a cDNA complementary to at least a portion of said RNA molecule.

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

Images