removing excess oligonucleotide from the reaction mixture

Abstract

The invention provides methods and compositions for removing unwanted or excess oligonucleotides from a reaction mixture using a double hairpin nucleic acid comprising a single nucleic acid strand having: i. a first hairpin at the 5' end; ii. a second hairpin at the 3' end; and iii. a single-stranded region between the 5' end and the 3' end, wherein the single-stranded region comprises a sequence capable of hybridizing to an oligonucleotide to be removed, such as an excess primer, subcode, or adaptor molecule.

Description

Technical Field

[0001] This invention relates to the field of nucleic acids. More specifically, this invention relates to the field of in vitro reactions involving nucleic acids. Background Technology

[0002] Reactions utilizing short-chain nucleic acids (“oligonucleotides”) typically end with an excess of oligonucleotides remaining in the reaction mixture. These oligonucleotides may hinder downstream processes through interactions with each other or with off-target sample nucleic acids. Such interactions deplete necessary reagents and prevent the desired reaction.

[0003] Excess oligonucleotides include primers, probes, and adaptors. For example, amplification reactions (including polymerase chain reaction (PCR) and ligation chain reaction (LCR)) utilize oligonucleotide primers and probes. Excess primers and probes not incorporated into the amplification product need to be removed before downstream processing.

[0004] Single-molecule massively parallel sequencing involves forming a nucleic acid library by attaching oligonucleotide adaptors with universal initiation binding sites and other essential features to the ends of each nucleic acid in the library. After library formation, excess adaptors not incorporated into the library nucleic acids need to be removed.

[0005] In the field of single-cell analysis, U.S. Patent 10,144,950 describes a novel single-cell analysis method called quantum barcode encoding (QBC), in which each cell is labeled with a unique combinatorial barcode. The combinatorial barcode is assembled from subcode oligonucleotides. At the end of the code assembly process, the quantum code needs to be removed.

[0006] Typically, removal involves multiple steps, including precipitation, centrifugation, or bead capture. These steps are time-consuming and reagent-intensive, and result in the loss of target nucleic acids separated from excess oligonucleotides. Therefore, a practical and economical method is needed to remove excess oligonucleotides from the reaction mixture. Summary of the Invention

[0007] This invention relates to a method for removing excess oligonucleotides from a reaction mixture by capturing and isolating them using a double-hairpin nucleic acid. The excess oligonucleotides are linked to a double-hairpin structure to form a topologically circular closed nucleic acid chain that does not interfere with downstream applications.

[0008] In some embodiments, the present invention is a method for removing unwanted oligonucleotides from a reaction mixture, the method comprising: contacting the reaction mixture with a double hairpin nucleic acid comprising a single nucleic acid strand having: a first hairpin at a 5′ end; a second hairpin at a 3′ end; and a single-stranded region between the 5′ and 3′ ends, wherein the single-stranded region contains a sequence capable of hybridizing with the oligonucleotide to be removed; annealing the oligonucleotide to be removed with the double hairpin nucleic acid; and ligating the oligonucleotide to be removed to the end of the double hairpin nucleic acid, thereby removing the unwanted oligonucleotide from the reaction mixture. The method further comprises contacting the reaction mixture with a ligase. In other embodiments, the reaction mixture already contains a ligase. In some embodiments, the double hairpin contains a 5′-phosphate group.

[0009] In some embodiments, the single-stranded region of the double hairpin oligonucleotide includes two regions complementary to the oligonucleotide to be removed, which are adjacent to a single intermediate region. This intermediate region may be a non-nucleotide spacer or a region containing inosine nucleotides. In some embodiments, the oligonucleotide to be removed comprises a plurality of oligonucleotides having two constant regions adjacent to a single barcode region that varies within the plurality of oligonucleotides.

[0010] In some embodiments, the present invention is a double-hairpin nucleic acid for capturing oligonucleotides from a reaction mixture, the double hairpin comprising a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends, wherein the single-stranded region contains a sequence capable of hybridizing with the oligonucleotide to be removed. The oligonucleotide may be composed of RNA or DNA. In some embodiments, the single-stranded region comprises two regions complementary to the oligonucleotide to be removed, the two regions being flanked by a single intermediate region. The intermediate region may be a non-nucleotide spacer or composed of inosine nucleotides.

[0011] In some embodiments, the present invention is a method for detecting multiple targets in multiple cells in a reaction mixture, the method comprising: binding to targets in multiple cells using a plurality of unique binding reagents, each of the plurality of unique binding reagents being specific for one of the targets; during successive multi-round split-cell synthesis, sequentially adding a plurality of code oligonucleotides to each of the binding reagents in the plurality of cells, wherein the code oligonucleotide in each round is annealed adjacent to a code oligonucleotide from the previous round via an annealing region, and the adjacent annealed code oligonucleotides are covalently linked to each other to form a unique cell-derived nucleotide code in each cell; removing the over-code oligonucleotide by contacting the reaction mixture with a double-hairpin nucleic acid comprising a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends, the single-stranded region being capable of hybridizing with the code oligonucleotide. The method may further comprise contacting the sample with a polynucleotide kinase. In some embodiments, the subcode oligonucleotide comprises a barcode region flanked by two annealing regions, and the single-stranded region of the double hairpin nucleic acid comprises a spacer of equal length to the barcode region flanked by two sequences capable of hybridizing with the annealing region in the subcode oligonucleotide. The spacer may be a carbon linker or may contain an inosine-containing nucleotide.

[0012] In some embodiments, the present invention is a method for preparing a solution of amplified target nucleic acid free of excess amplification primers, the method comprising: contacting a reaction mixture containing target nucleic acid with forward and reverse amplification primers and a thermostable nucleic acid polymerase in the presence of a reagent supporting nucleic acid synthesis; subjecting the reaction mixture to a thermal cycling treatment suitable for annealing and extending the forward and reverse primers; after thermal cycling, contacting the reaction mixture with a nucleic acid ligase and a double-hairpin nucleic acid composed of a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends, wherein the single-stranded region is capable of hybridizing with either the forward or reverse primer; ligating the forward and reverse primers to the corresponding double-hairpin nucleic acid, thereby removing the forward and reverse primers from the solution of amplified target nucleic acid. The method may further include contacting the sample with a polynucleotide kinase.

[0013] In some embodiments, the present invention is a method for preparing a solution of amplified target nucleic acid without excess amplification primers, the method comprising: contacting a reaction mixture containing the target nucleic acid with a first probe and a second probe capable of hybridizing adjacent to the target nucleic acid and a thermostable ligase in the presence of a ligation-supporting reagent; subjecting the reaction mixture to a thermal cycling treatment suitable for annealing and ligating the first probe and the second probe to each other; after thermal cycling, contacting the reaction mixture with a double hairpin nucleic acid composed of a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends, wherein the single-stranded region is capable of hybridizing with either the first probe or the second probe; ligating the first probe and the second probe to the corresponding double hairpin nucleic acid, thereby removing the first probe and the second probe from the solution of amplified target nucleic acid. The method may further include contacting the sample with a polynucleotide kinase.

[0014] In some embodiments, the present invention is a method for forming a nucleic acid library free of excess adaptor molecules, the method comprising: contacting a reaction mixture containing nucleic acids with an adaptor molecule and a ligase; ligating the adaptor molecule to the end of the nucleic acid; contacting the reaction mixture with a double-hairpin nucleic acid composed of a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends, wherein the single-stranded region is capable of hybridizing with the adaptor; and ligating the adaptor to the double-hairpin nucleic acid, thereby removing the adaptor from a solution of amplified target nucleic acid. In some embodiments, the adaptor consists of two oligonucleotides formed in at least one double-stranded region. In some embodiments, the reaction mixture is subjected to an elevated temperature sufficient to separate the adaptor into single strands. In some embodiments, the double-hairpin nucleic acid comprises a mixture of two double-hairpin nucleic acids, each having a single-stranded region complementary to one strand of the adaptor. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the double hair clip structure and its usage. Detailed Implementation

[0016] The term “nucleic acid” refers to a polymer of nucleotides, and unless otherwise limited, it also includes known natural nucleotide analogs that are capable of functioning (e.g., hybridizing) with naturally occurring nucleotides in a similar manner.

[0017] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably. They refer to nucleotides of any length in polymeric form, being deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include: coding or non-coding regions of genes or gene segments; intergenic DNA; loci defined by bond-binding analysis; exons; introns; messenger RNA (mRNA); transfer RNA; ribosomal RNA; short interfering RNA (siRNA); short hairpin RNA (shRNA); microRNA (miRNA); small nucleolar RNA; ribozymes; complementary DNA (cDNA), which is the DNA expression of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; synthetic or amplified DNA molecules; genomic DNA; recombinant polynucleotides; branched polynucleotides; plasmids; vectors; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probes; and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Unless otherwise specified, polynucleotide sequences are listed in a 5′ to 3′ orientation when provided. The term "oligonucleotide" refers to a shorter nucleic acid, although longer nucleic acids can also be called oligonucleotides, but their length is generally no more than 100 nucleotides.

[0018] A nucleic acid "probe" is an oligonucleotide that binds to a target nucleic acid with a complementary sequence via one or more types of chemical bonds, typically through complementary base pairing, usually formed by hydrogen bonds, resulting in a double-stranded structure. The probe binds to or hybridizes with a "probe binding site." Probes can be labeled with detectable tags to allow for simple detection, especially when the probe hybridizes to its complementary target. Alternatively, probes can be unlabeled but can still be detected by specific binding directly or indirectly to a labeled ligand.

[0019] The terms “epitope” and “target molecule” are used interchangeably in this document and refer to the target molecule (protein or nucleic acid) that is detected and / or quantified by the methods described herein.

[0020] The term "target oligonucleotide" refers to an oligonucleotide that has been removed or depleted from a reaction mixture using the novel methods disclosed herein. A target oligonucleotide may share similarities with the target molecule as defined above (e.g., sharing some nucleic acid sequences), but "target oligonucleotide" refers only to a target oligonucleotide removed from the reaction mixture according to the methods disclosed herein. Target oligonucleotides are interchangeably referred to as "excess oligonucleotide," "unwanted oligonucleotide," "oligonucleotide to be depleted," and "oligonucleotide to be removed."

[0021] The term "hairpin" in nucleic acid terminology refers to a secondary structure formed by a single strand of nucleic acid, which has at least complementary regions that are capable of annealing each other. A hairpin includes a short intermediate region between two complementary regions, causing the nucleic acid strand to bend sharply during annealing. Hairpins with longer intermediate regions do not form sharp bends but rather loops, and this structure can be called a "stem-loop" structure. There is no precise boundary between a hairpin and a stem-loop. Stem-loop structures with smaller loops are sometimes called hairpins.

[0022] This invention provides an improved method for removing excess or unwanted oligonucleotides from a reaction mixture without any purification steps. This method can easily remove or inactivate oligonucleotides with minimal or no operation time and without loss of the desired product.

[0023] In reactions involving mixtures of oligonucleotides, controlling and eliminating byproducts and excess reagents is crucial, as they can act as substrates in troublesome side reactions. Side reactions such as primer-primer or probe-probe interactions, off-target initiation, and off-target probe binding can affect the kinetics of the intended reaction by isolating reactants or consuming reactant components. The present invention disclosed herein includes methods and compositions for rendering specific oligonucleotide sequences inert to side reactions and off-target base pairing.

[0024] In some embodiments, the invention comprises short oligonucleotides (DNA or RNA), referred herein as "double hairpins" (…). Figure 1 After the reaction involving the removal of unused excess oligonucleotides is completed, a double hairpin oligonucleotide is added to the reaction mixture. The sequence of the double hairpin oligonucleotide is designed such that it: 1) forms a hairpin at each end, and 2) has an intermediate portion between the hairpins that binds to the oligonucleotide to be removed or to exhibit inertness (“target oligonucleotide”). When the double hairpin oligonucleotide is added to the mixture containing the target oligonucleotide, the target oligonucleotide anneals to the intermediate portion of the double hairpin. Upon annealing, a ligase is used to form covalent bonds between the 5′ and 3′ ends of the double hairpin oligonucleotide and the 5′ and 3′ ends of the target oligonucleotide, thereby forming a topologically cyclic inert product.

[0025] This cyclic product renders the target oligonucleotide inert in several ways. First, the ligation blocks the 3′ end of the target oligonucleotide, serving as a primer in any primer extension reaction, including exponential amplification. Second, the bases pair with the double hairpin oligonucleotide, preventing the target oligonucleotide from pairing with other nucleic acids in the reaction mixture.

[0026] This invention relates to a method for manipulating nucleic acids in a sample. In some embodiments, the sample is obtained from a subject or patient. In some embodiments, the sample may include fragments of solid tissue or solid tumors obtained, for example, by biopsy from the subject or patient. The sample may also include bodily fluids (e.g., urine, sputum, serum, plasma or lymph, saliva, phlegm, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid, or fecal samples) that may contain nucleic acids. The sample may include whole blood or a blood fraction in which nucleic acids may be present. In other embodiments, the sample is a cultured sample, such as a tissue culture containing cells from which nucleic acids can be isolated. In some embodiments, the target nucleic acid in the sample is derived from an infectious agent, such as a virus, bacteria, protozoa, or fungus. In some embodiments, sample cells are used in a procedure involving the removal of target oligonucleotides. In other embodiments, nucleic acids isolated from the sample are used.

[0027] In some embodiments, it is necessary to isolate nucleic acids from a sample. DNA extraction methods are known in the art. See J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, 2nd ed., ColdSpring Harbor Laboratory Press: New York, NY. Various kits are commercially available for the extraction of nucleic acids (DNA or RNA) from biological samples (e.g., BD Biosciences Clontech (Palo Alto, Cal.); Epicentre Technologies (Madison, Wisc.); Gentra Systems, INC. (Minneapolis, Minn.); and Qiagen, INC. (Valencia, Cal.); Ambion, Inc. (Austin, Tex.); BioRad Laboratories (Hercules, Cal.); etc.).

[0028] In some embodiments, purification is performed via affinity binding. In a variation of this embodiment, the affinity is targeted at a specific target sequence (sequence capture). In other embodiments, the primers contain affinity tags. Any affinity tag known in the art can be used, such as biotin, antibodies, or antigens in which specific antibodies are present. The affinity pair of the affinity tag can be present in solution, for example, on a solid support (such as suspended particles or beads), or bound to a solid support.

[0029] In some embodiments, size exclusion chromatography, electrophoresis, or accelerated electrophoresis are used to separate nucleic acids by size and purify them.

[0030] Nucleic acids, proteins, or other target biomarkers can be present in cells or reaction mixtures and can serve as targets for detection or quantification procedures. As disclosed herein, detection or quantification procedures are improved by removing excess oligonucleotides.

[0031] Each nucleic acid target is characterized by its nucleic acid sequence. Each protein target is characterized by its amino acid sequence and its epitope recognized by a specific antibody. In some embodiments, the target nucleic acid contains a locus of a genetic variant, such as an addition, a polymorphism, including single nucleotide polymorphisms or variants (SNPs of SNVs), or a gene rearrangement that results in, for example, gene fusion. In some embodiments, the protein biomarker contains amino acid changes that result in the formation of a unique epitope. In some embodiments, the target nucleic acid or target protein contains a biomarker, i.e., a gene or protein antigen, a variant of which is associated with a disease or condition. For example, the target nucleic acid or target protein may be selected from a combination of disease-related biomarkers described in U.S. Patent Application Serial No. 14 / 774,518, filed September 10, 2015. Such combinations are available as the AVENIO ctDNA Analysis Kit (Roche Sequencing Solutions, Pleasanton, Cal). In other embodiments, the target nucleic acid or target protein is characterized by a specific organism and helps to identify the organism, or is characterized by a pathogenic organism, such as drug sensitivity or resistance. In other embodiments, the target nucleic acid or target protein has unique characteristics of the human subject, such as a combination of HLA or KIR sequences defining the subject's unique HLA or KIR genotype. In other embodiments, the target nucleic acid is a somatic cell sequence, such as a rearranged immune sequence representing an immunoglobulin (including IgG, IgM, and IgA immunoglobulins) or a T-cell receptor (TCR) sequence. In yet another application, the target is a fetal sequence present in the mother's blood, including fetal sequences characteristic of fetal diseases or conditions or pregnancy-related maternal conditions. For example, the target may be one or more autosomal or X-linked diseases described by Zhang et al. ((2019) Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA, Nature Med. 25(3): 439).

[0032] In some embodiments, the target is a nucleic acid (including mRNA, microRNA, viral RNA, cellular DNA, or cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA)).

[0033] In some embodiments, the target is a protein expressed in the cell. For example, the protein target can be a cell surface protein. In some embodiments, the cell surface protein is a lymphocyte surface protein selected from inhibitory receptors (such as Pdcd1, Havrcr2, Lag3, CD244, Entpd1, CD38, CD101, Tigit, CTLA4), cell surface receptors (such as TNFRSF9, TNFRSF4, Klrg1, CD28, Icos, IL2Rb, IL7R), or cytokine receptors (such as CX3CR1, CCL5, CCL4, CCL3, CSF1, CXCR5, CCR7, XCL1, and CXCL10). In some embodiments, the protein is selected from CD4, CD8, CD11, CD16, CD19, CD20, CD45, CD56, and CD279.

[0034] refer to Figure 1 The present invention includes a novel composition comprising a double hairpin oligonucleotide (also known as a double hairpin nucleic acid) for capturing unwanted oligonucleotides from a reaction mixture. The oligonucleotide comprises three parts: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ hairpins, wherein the single-stranded region is capable of hybridizing with the oligonucleotide to be removed (“target oligonucleotide”).

[0035] Each hairpin is formed by two sequences within an oligonucleotide that are capable of forming a stable hybrid with each other and creating a DNA (or RNA) bend between the hybrid sequences. In some embodiments, one or two hairpins are formed by regions that are completely complementary to each other. In other embodiments, one or two hairpins are formed by only partially complementary sequences; however, they form a stable hairpin.

[0036] In some embodiments, one or both hairpin regions of the double hairpin oligonucleotide contain one or more modified nucleotides to improve the thermal stability (melting temperature, Tm) of the hairpin structure. For example, one or both hairpin regions include one or more of 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutyn-2′-deoxyuridine, 8-aza-7-deazoguanylic acid, ribonucleotide, 2′O-methylribonucleotide, or locked nucleic acid.

[0037] In some embodiments, the hairpin-forming sequence is an artificial sequence, optionally a computer-designed nucleic acid sequence. In other embodiments, the hairpin-forming sequence is a naturally occurring hairpin-forming sequence, which is reviewed in the following literature: Bikard et al. (2010) Folded DNA in Action: Hairpin Formation and Biological Functions in Prokaryotes, Microbiol. Mol. Biol. Review 74(4): 570-588; or Brazda et al. (2011) Cruciform structures are a common DNA feature important for regulating biological process, BMC Mol. Biol. 12: 33.

[0038] The single-stranded portion of the hairpin is designed to form a stable hybrid with the target oligonucleotide, allowing the target oligonucleotide to attach to both ends of the double hairpin oligonucleotide. Those skilled in the art will understand that the stable hybrid can be formed from nucleic acid strands that are both perfectly complementary and imperfectly complementary. In some embodiments, the single-stranded region of the double hairpin oligonucleotide is partially complementary to the target oligonucleotide. In some embodiments, the single-stranded region of the double hairpin oligonucleotide is perfectly complementary to the target oligonucleotide. In some embodiments, a portion of the single-stranded region of the double hairpin oligonucleotide is perfectly complementary to the target oligonucleotide. In some embodiments, the 5′ and 3′ adjacent portions of the single-stranded region are perfectly complementary to the 3′ and 5′ adjacent portions of the target oligonucleotide, while the middle portion of the single-stranded region is not complementary or only partially complementary to the middle portion of the target oligonucleotide.

[0039] In some embodiments, the length of the unfolded double hairpin oligonucleotide is between about 40 and about 200 nucleotides. The length of the hairpin-forming sequence is between about 9 and about 40 nucleotides. While there is no prescribed length for either of the two hairpin regions, it is desirable that the lengths of both hairpin regions be sufficient to ensure the stability of the hairpin in the reaction mixture. Those skilled in the art can utilize various tools to predict the stability (unwinding temperature, Tm) of the nucleic acid duplex under various temperature and salt conditions. The single-stranded region is tailored to fit the target oligonucleotide. The oligonucleotide includes primers, probes, and adaptors, and its length is typically between 10 and 100 nucleotides, with most being between 20 and 50 nucleotides. Therefore, the length of the single-stranded region of the double hairpin oligonucleotide is in the range of about 10 to about 100 nucleotides, with its length most commonly between 20 and 50 nucleotides.

[0040] In some embodiments, the present invention is a method for removing excess or unwanted oligonucleotides (“target oligonucleotides”) from a reaction mixture. The method includes contacting a reaction mixture containing the target oligonucleotides with a double-hairpin nucleic acid comprising a single nucleic acid strand having: a first hairpin at the 5′ end; a second hairpin at the 3′ end; and a single-stranded region between the 5′ and 3′ ends. The single-stranded region contains a sequence capable of hybridizing with the target oligonucleotide.

[0041] A double hairpin oligonucleotide is added at a concentration calculated to exceed the concentration of the target oligonucleotide to be removed, to ensure favorable hybridization kinetics. Those skilled in the art will understand that in some cases, the optimal molar concentration of the double hairpin oligonucleotide can be calculated in advance, while in others, it is determined experimentally by titrating the double hairpin oligonucleotide. A significant advantage of this invention is that the double hairpin oligonucleotide is designed to interact with the target oligonucleotide without interacting with any other nucleic acids in the reaction mixture.

[0042] In some embodiments, the reaction mixture is incubated at the optimal temperature for forming a hybrid between the target oligonucleotide and the double hairpin oligonucleotide. In some embodiments, the annealing temperature is in the range of 40°C to 72°C. In some embodiments, one or both of the hybridization and ligation steps occur at ambient temperature.

[0043] In some embodiments, the reaction is incubated for the time required for hybridization to form between the target oligonucleotide and the double hairpin oligonucleotide. In some embodiments, this time is 5 to 15 minutes.

[0044] In some embodiments, a single target oligonucleotide is removed and a single double hairpin oligonucleotide is added. In other embodiments, a mixture of multiple target oligonucleotides is removed and multiple double hairpin oligonucleotides are added.

[0045] After annealing the single-stranded regions of the target oligonucleotide and the double hairpin oligonucleotide, the reaction mixture is contacted with a ligase. A ligase is available for simultaneously ligating DNA and RNA, and will be adapted to both the DNA and RNA double hairpin oligonucleotides and the target oligonucleotide. A ligase suitable for the nucleic acid type in both the double hairpin oligonucleotide and the target oligonucleotide is used. Both ends of the target oligonucleotide are ligated to the double hairpin oligonucleotide. Figure 1 ).

[0046] In some embodiments, the ligase and ligase cofactor, along with the substrate (e.g., ATP), are present in the reaction mixture because the upstream process has already occurred. In such embodiments, no additional ligase or ligase cofactor is added to remove the target oligonucleotide. In other embodiments, the ligase and ligase cofactor are added.

[0047] In some embodiments, both the target oligonucleotide and the double hairpin oligonucleotide contain a 5′-phosphate group. In other embodiments, one or both of the target oligonucleotide and the double hairpin oligonucleotide lack a 5′-phosphate group. In such embodiments, the reaction mixture is contacted with a polynucleotide kinase (PNK) and any necessary cofactors (e.g., ATP), and the 5′ end is phosphorylated to allow the linkage to occur.

[0048] In some embodiments, enzymes (e.g., ligases and polynucleotide kinases) are removed or inactivated (e.g., heat-inactivated) prior to downstream steps involving the depletion of the target oligonucleotide by the reaction mixture.

[0049] Those skilled in the art will understand that the term "removal" regarding target oligonucleotides encompasses all forms of inactivation of the target oligonucleotide. If the target oligonucleotide is no longer able to hybridize with any nucleic acid in the reaction mixture and can no longer initiate nucleic acid synthesis, the oligonucleotide is effectively removed from the reaction mixture. Therefore, the target oligonucleotide is isolated within a double-stranded, covalently closed, topologically circular nucleic acid formed by the double hairpin oligonucleotide and the target oligonucleotide. Figure 1 (The bottom) constitutes the removal of target oligonucleotides from the reaction mixture.

[0050] In some embodiments, the present invention is an improved method for nucleic acid amplification by polymerase chain reaction (PCR). As detailed in U.S. Patent No. 4,683,195, polymerase chain reaction (PCR) involves contacting a sample solution containing nucleic acids with forward primers, reverse primers, a nucleic acid polymerase (preferably a thermostable polymerase), and a nucleic acid precursor (such as dNTPs or dNTP analogs that can be incorporated by the nucleic acid polymerase). The reaction mixture is subjected to a thermal cycling process comprising multiple cycles, including a DNA denaturation step (90°C or higher), an optional primer annealing step (45°C to 72°C), and a polymerase extension step (65°C to 72°C). In some embodiments, the PCR is real-time PCR, including fluorescently labeled detection probes, as described in U.S. Patent No. 5,804,375. In prior art, after the amplification reaction is complete, the reaction mixture is subjected to a purification step (such as SPRI bead purification) to remove excess primers. The present invention eliminates the purification step.

[0051] In some embodiments, after the amplification reaction is complete, the reaction mixture is contacted with the novel double-hairpin oligonucleotide described herein. In some embodiments, the target oligonucleotide is a mixture of a forward primer and a reverse primer. In some embodiments, the target oligonucleotide mixture further includes a detection probe. The double-hairpin oligonucleotide is a combination of two or three oligonucleotides. The first double-hairpin oligonucleotide includes an intermediate region complementary to the forward amplification primer. The second double-hairpin oligonucleotide includes an intermediate region complementary to the reverse amplification primer. A third double-hairpin oligonucleotide targeting the detection probe may also be present. The third double-hairpin oligonucleotide includes an intermediate region complementary to the detection probe. Excess primers and probes (if present) are ligated to the corresponding double-hairpin oligonucleotides to form a closed loop structure.

[0052] In some embodiments, prior to ligation, the reaction mixture is contacted with a polynucleotide kinase (PNK) and any necessary cofactors and substrates (e.g., ATP) to phosphorylate the 5′ ends of the forward and reverse primers and (if present) the probes, thereby enabling ligation to occur.

[0053] In some embodiments, the double hairpin oligonucleotide is used for all three target oligonucleotides: the forward primer, the reverse primer, and the probe. In other embodiments, the double hairpin oligonucleotide is used for only one or two of the three target oligonucleotides.

[0054] After the excess primers and (if present) excess probes are isolated in a closed loop structure, downstream steps can be performed immediately.

[0055] In some embodiments, the present invention is an improved method for nucleic acid amplification via ligase chain reaction (LCR). As detailed in U.S. Patent No. 6,312,892, ligase chain reaction (LCR) involves contacting a sample solution containing nucleic acids with a probe set (probes capable of hybridizing adjacently to a nucleic acid template) containing a first oligonucleotide probe and a second oligonucleotide probe, and a ligase (preferably a thermostable ligase). The reaction mixture is subjected to a thermal cycling process comprising multiple cycles including a DNA denaturation step (90°C or higher), an optional probe annealing step (45°C to 65°C), and a ligation step (65°C or lower). In prior art, after the amplification reaction is complete, the reaction mixture is subjected to a purification step (e.g., SPRI bead purification) to remove excess probe. The present invention eliminates the purification step.

[0056] In some embodiments, after the amplification reaction is complete, the reaction mixture is contacted with the novel double hairpin oligonucleotide described herein. In some embodiments, the target oligonucleotide is a mixture of a first probe and a second probe. The double hairpin oligonucleotide is a combination of two oligonucleotides. The first double hairpin oligonucleotide includes an intermediate region complementary to the first probe. The second double hairpin oligonucleotide includes an intermediate region complementary to the second probe. Conveniently, the ligase and all necessary cofactors are already present in the reaction mixture. An excess probe is ligated to the corresponding double hairpin oligonucleotide to form a closed loop structure.

[0057] In some embodiments, prior to ligation, the reaction mixture is contacted with a polynucleotide kinase (PNK) and any necessary cofactors and substrates (e.g., ATP) to phosphorylate the 5' end of the probe, enabling ligation to occur.

[0058] In some embodiments, the double hairpin oligonucleotide is used for both probes. In other embodiments, the double hairpin oligonucleotide is used for only one of the two probes.

[0059] After the excess primers and (if present) probes are isolated in a closed loop structure, downstream steps can be performed immediately.

[0060] In some embodiments, the present invention is an improved method for performing quantum barcode encoding (QBC) (a method for detecting multiple targets in multiple single cells, as described in U.S. Patent 10,144,950). The method includes preparing a cell suspension. Next, the cells are contacted with a unique binding agent (e.g., a DNA probe or RNA probe (including aptamers) or an antibody). The probe or antibody contains at least one portion or element that specifically interacts with the target and elements that allow the assembly of combined nucleic acid barcodes. In some embodiments, the assay is a multiplex assay, wherein multiple target molecules in multiple cells in a single reaction mixture are detected using multiple different binding agents of the same or different kinds (e.g., multiple different nucleic acid probes, or multiple different antibodies, or a combination of nucleic acid probes and antibodies). If the unique binding agent is in the antibody, the antibody may contain a linker oligonucleotide that facilitates the assembly of the barcode. Methods for attaching nucleic acids to antibodies are known, for example: Gullberg et al., PNAS 101(22), pp. 228420-8424 (2004); Boozer et al., Analytical Chemistry, 76(23), pp. 6967-6972 (2004); or Kozlov et al., Biopolymers 5:73(5), pp. 621-630 (2004). If the unique binding agent is a nucleic acid probe, the barcode can be attached directly to the probe.

[0061] Cells with binding probes or antibodies are assembled using cleavage barcodes after a unique binding agent binds to its target (as detailed in U.S. Patent 10,144,950, which is incorporated herein by reference). A unique cell-derived code is assembled onto each cell in which the unique binding agent has been bound. The unique cell barcode is a modular structure assembled from subunits through stepwise addition of subunits. Each subunit contains a barcode and an attachment region for linking the subunit to the growing unique cell barcode. Subunits are linked to each other or to a universal backbone via the attachment region (e.g., a complementary nucleic acid sequence). The attachment region may contain one or both of hybridization with the backbone or hybridization with an adjacent subunit and linking with an adjacent subunit. After the unique cell barcodes have been assembled, they are isolated from the cells and detected. For example, the unique cell barcodes are amplified and sequenced.

[0062] In one embodiment of the invention, prior to the amplification step, a reaction mixture containing a unique barcode is contacted with the novel double-hairpin oligonucleotide described herein. The target oligonucleotide is a mixture of multiple barcode subunits. The double-hairpin oligonucleotide is designed to bind one or more (including all) of the barcode subunits in the mixture. In some embodiments, the single-stranded portion of the double-hairpin oligonucleotide includes two regions capable of hybridizing with annealed regions of the barcode subunits, these two regions being flanked by a single intermediate region having the length of a subcode contained in the barcode subunit. Because the subcode varies in the barcode subunits, the intermediate region of the double-hairpin oligonucleotide is designed to accommodate this diversity. In some embodiments, the intermediate region of the double-hairpin oligonucleotide is a non-nucleotide spacer with a length corresponding to the length of the subcode, or is inosine containing nucleic acids (the number of inosine nucleotides corresponds to the number of nucleotides in the subcode).

[0063] In some embodiments, the barcode subunit oligonucleotides can be linked into unique barcodes, i.e., conveniently already containing 5′-phosphate esters. Furthermore, in such embodiments, an active ligase is conveniently present in the reaction mixture. In such embodiments, the reaction mixture is contacted only with the double hairpin oligonucleotides.

[0064] In other embodiments, the barcode subunit is incorporated into a unique barcode using a method different from ligation (see, for example, an embodiment where the barcode is copied by polymerase, see U.S. Patent Application Serial No. 16 / 250,974, filed January 19, 2019). In such embodiments, the reaction mixture is contacted with a double hairpin oligonucleotide and further with a ligase. If the barcode subunit lacks a 5′-phosphate, the reaction mixture is further contacted with a polynucleotide kinase (PNK). In some embodiments, the reaction mixture is contacted with ATP (a cofactor required for both the kinase and the ligase).

[0065] Excess barcode subunits are linked to double-hairpin oligonucleotides to form a closed circular structure. Amplification can then proceed immediately after the excess barcode subunit oligonucleotides are isolated within this closed circular structure. The barcode oligonucleotides can no longer interfere with nucleic acid amplification, for example, by being used as amplification primers.

[0066] Of particular advantage is that, with this invention, no purification step is required prior to amplification. Quantum barcode encoding (QBC) is a single-cell analysis method in which each unique cell barcode represents a single cell. Therefore, the loss of a single unique cell barcode molecule represents the loss of valuable data points. With the method described herein, downstream analysis steps can be performed immediately after removing excess barcode subunits.

[0067] In some embodiments, the present invention is an improved method for forming a library for nucleic acid sequencing. In this embodiment, nucleic acids in a sample are ligated to adapters, and excess adapters are removed before downstream library processing.

[0068] In some embodiments, the method includes treating the nucleic acids in the sample prior to ligating the adaptor. The sample nucleic acids may already be blunt-ended or may be endowed with blunt ends through enzymatic treatment (e.g., “end repair”). In other embodiments, the blunt-ended DNA undergoes alpha tailing, where a single A nucleotide is added to the 3′ end of one or both blunt ends. The adaptor may be a double-stranded or partially double-stranded short nucleic acid having a blunt end from which a single T nucleotide extends to facilitate ligation between the sample nucleic acid and the adaptor. Commercially available kits for performing end repair, alpha tailing, and adaptor ligation include the AVENIO ctDNA library preparation kit or the KAPA HyperPrep and HyperPlus kit (Roche Sequencing Solutions, Pleasanton, Cal.).

[0069] In some embodiments, the invention includes a novel step of removing excess adaptor from the library formation reaction by isolating excess adaptor within a closed circular structure formed by linking it to the double hairpin oligonucleotides described herein. The target oligonucleotide comprises two strands of the adaptor. A double hairpin is a combination of two double hairpin oligonucleotides. The first double hairpin oligonucleotide contains an intermediate region complementary to the first strand of the adaptor, while the second double hairpin oligonucleotide contains an intermediate region complementary to the second strand of the adaptor.

[0070] In some embodiments, the method further includes the step of incubating a reaction mixture containing the library and an excess of the adaptor at an elevated temperature sufficient to separate the strand of the adaptor but insufficient to separate the hairpin structure in the library nucleic acid or the double hairpin oligonucleotide. To facilitate this step of the method, the double hairpin oligonucleotide may contain a modified nucleotide that increases the melting temperature (Tm) of the hairpin. For example, one or both hairpin moieties comprise one or more of 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutyn-2′-deoxyuridine, 8-aza-7-deazoguanylic acid, ribonucleotide, 2′O-methylribonucleotide, or locked nucleic acid.

[0071] In some embodiments, the reaction mixture is also contacted with a polynucleotide kinase to phosphorylate the 5′ end of the adaptor.

[0072] The method of the present invention further includes the step of linking an adapter strand to a double hairpin oligonucleotide. The method may further include the steps of amplifying the library or subjecting the library to target capture. No purification step is required prior to the downstream amplification or target capture step.

[0073] The method for removing excess oligonucleotides of the present invention is not limited to the exemplary applications specifically described above, but can be used in any diagnostic, prognostic, therapeutic, patient stratification, drug development, treatment selection, and screening process involving the use of oligonucleotides and in which the removal of excess oligonucleotides is required.

Claims

1. A method for removing unwanted oligonucleotides from a reaction mixture, the method comprising: a. Contact the reaction mixture with a double hairpin nucleic acid comprising a single nucleic acid chain, the single nucleic acid chain having: i. The first hairpin at the 5' end; ii. The second hairpin at the 3' end; and iii. A single-stranded region between the 5' end and the 3' end, wherein the single-stranded region contains a sequence capable of hybridizing with the oligonucleotide to be removed; b. Anneal the oligonucleotide to be removed with the double hairpin nucleic acid; c. The oligonucleotide to be removed is ligated to the end of the double hairpin nucleic acid to form an inert cyclic product, thereby removing the unwanted oligonucleotide from the reaction mixture.

2. The method of claim 1, further comprising contacting the reaction mixture with a ligase prior to step c.

3. The method of claim 1, wherein the reaction mixture comprises a ligase prior to step a.

4. The method of claim 1, wherein the dual hairpins contain a 5'-phosphate group.

5. The method of claim 1, wherein the single-stranded region comprises two regions complementary to the oligonucleotide to be removed, the two regions being adjacent to a single intermediate region.

6. The method of claim 5, wherein the intermediate region is a non-nucleotide spacer.

7. The method of claim 5, wherein the intermediate region comprises inosine nucleotides.

8. The method of claim 5, wherein the oligonucleotide to be removed comprises a plurality of oligonucleotides having two constant regions adjacent to a single barcode region that is distinct among the plurality of oligonucleotides.

9. A method for detecting multiple targets in multiple cells of a reaction mixture, the method comprising: a. Binding a plurality of unique binding agents to the target in a plurality of cells, each of the plurality of unique binding agents being specific to one of the targets; b. During successive rounds of split-cell synthesis, a plurality of subcode oligonucleotides are added in an orderly manner to each of the binding reagents in the plurality of cells, wherein the subcode oligonucleotide in each round is annealed adjacent to the subcode oligonucleotide from the previous round via an annealing zone, and the adjacent annealed subcode oligonucleotides are covalently linked to each other to form a unique cell-derived nucleotide code in each cell. c. Contact the reaction mixture with a double hairpin nucleic acid comprising a single nucleic acid strand having: a first hairpin at the 5' end; a second hairpin at the 3' end; and a single-stranded region between the 5' and 3' ends, the single-stranded region being capable of hybridizing with the code oligonucleotide and linking the over-code oligonucleotide to the corresponding double hairpin nucleic acid to form an inert cyclic product, thereby removing the over-code oligonucleotide.

10. The method of claim 9, wherein the subcode oligonucleotide comprises a barcode region side-attached by two annealing regions, and the single-stranded region of the double hairpin nucleic acid comprises a spacer of equal length to the barcode region, the spacer being side-attached by two sequences capable of hybridizing with the annealing region in the subcode oligonucleotide.

11. A method for preparing a solution of amplified target nucleic acid without excess amplification primers, the method comprising: a. In the presence of reagents supporting nucleic acid synthesis, contact the reaction mixture containing the target nucleic acid with forward and reverse amplification primers and a thermostable nucleic acid polymerase; b. Subject the reaction mixture to annealing suitable for the forward and reverse primers and to extended thermal cycling spectra; c. After completing the thermal cycling spectrum, the reaction mixture is contacted with a nuclease and a double hairpin nucleic acid composed of a single nucleic acid chain having: i. The first hairpin at the 5' end; ii. The second hairpin at the 3' end; and iii. A single-stranded region between the 5' end and the 3' end, wherein the single-stranded region is capable of hybridizing with the forward primer or with the reverse primer; d. Link the forward primer and the reverse primer to the corresponding hairpin nucleic acid to form an inert circular product, thereby removing the forward primer and the reverse primer from the solution of the amplified target nucleic acid.

12. A method for preparing a solution of amplified target nucleic acid without excess probe, the method comprising: a. In the presence of a ligation-supporting reagent, a reaction mixture containing the target nucleic acid is contacted with a first probe and a second probe capable of hybridizing adjacent to the target nucleic acid, as well as a heat-stable ligase; b. subject the reaction mixture to annealing suitable for the first probe and the second probe, as well as thermal cycling spectra of the interconnected components; c. After completing the thermal cycling spectrum, the reaction mixture is contacted with a double hairpin nucleic acid composed of a single nucleic acid chain, the single nucleic acid chain having: i. The first hairpin at the 5' end; ii. The second hairpin at the 3' end; and iii. A single-stranded region between the 5' end and the 3' end, wherein the single-stranded region is capable of hybridizing with the first probe or with the second probe; d. Link the first probe and the second probe to the corresponding double hairpin nucleic acid to form an inert circular product, thereby removing the first probe and the second probe from the solution of the amplified target nucleic acid.

13. A method for forming a library of nucleic acids free of excess adaptor molecules, the method comprising: a. Contact the reaction mixture containing nucleic acids with the adaptor molecules and ligase; b. Linking the adaptor molecule to the end of the nucleic acid to form a nucleic acid library; c. Contact the reaction mixture with a double hairpin nucleic acid composed of a single nucleic acid chain, the single nucleic acid chain having: i. The first hairpin at the 5' end; ii. The second hairpin at the 3' end; and iii. A single-stranded region between the 5' end and the 3' end, wherein the single-stranded region is capable of hybridizing with the adaptor; d. The adapter is ligated to the double hairpin nucleic acid to form an inert circular product, thereby removing the adapter from the solution containing the nucleic acid library.

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