Removal of excess oligonucleotide from the reaction mixture

Abstract

The present 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 that can hybridize to the oligonucleotide to be removed, e.g., excess primer, subcode, or adapter molecules.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of nucleic acids. More specifically, the present invention relates to the field of in vitro reactions involving nucleic acids. [Background technology]

[0002] 2. Background of the Invention Reactions utilizing short strands of nucleic acid ("oligonucleotides") often end up with excess oligonucleotides remaining in the reaction mixture. These oligonucleotides can interfere with downstream processes by interacting with each other or with off-target sample nucleic acids. Such interactions can deplete essential reagents and prevent the desired reaction.

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

[0004] Single molecule massively parallel sequencing involves forming a nucleic acid library by attaching an oligonucleotide adapter molecule having a universal priming binding site and other required characteristics to the end of each nucleic acid in the library. After the library is formed, it is necessary to remove excess adapter molecules that are not incorporated into the library nucleic acids.

[0005] In the field of single-cell analysis, US Patent No. 10,144,950 describes a novel process of single-cell analysis called quantum barcoding (QBC), in which each cell is labeled with a unique combinatorial barcode. The combinatorial barcode is constructed from subcode oligonucleotides. At the end of the code assembly process, the excess subcodes need to be removed.

[0006] Typically, removal involves a multi-step process, including precipitation, centrifugation or bead capture. These steps consume time and reagents, and cause loss of target nucleic acid that is separated from excess oligonucleotide. Therefore, there is a need for a practical and economical method to remove excess oligonucleotide from a reaction mixture. Summary of the Invention

[0007] Summary of the Invention The present invention is a method for removing excess oligonucleotides from a reaction mixture by capturing and sequestering them with a double hairpin nucleic acid, where the excess oligonucleotides are ligated into the double hairpin structure to form a topologically circular closed nucleic acid strand that does not interfere with downstream applications.

[0008] In some embodiments, the present invention is a method for removing undesired oligonucleotides from a reaction mixture, comprising 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'-end and the 3'-end, the single-stranded region comprising a sequence capable of hybridizing to the oligonucleotide to be removed, annealing the oligonucleotide to be removed to the double hairpin nucleic acid, and ligating the oligonucleotide to be removed to the end of the double hairpin nucleic acid, thereby removing the undesired oligonucleotides from the reaction mixture. The method may further comprise contacting the reaction mixture with a ligase. In other embodiments, the reaction mixture already comprises a ligase. In some embodiments, the double hairpin nucleic acid comprises a 5'-phosphate group.

[0009] In some embodiments, the single-stranded region of the double hairpin oligonucleotide comprises two regions of complementarity to the oligonucleotide to be removed, adjacent to a single intermediate region.The intermediate region can be a region that comprises a non-nucleotide spacer or an inosine nucleotide.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 between the plurality of oligonucleotides.

[0010] In some embodiments, the present invention is a double hairpin nucleic acid for capturing an oligonucleotide from a reaction mixture, the double hairpin nucleic acid comprises 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'-end and the 3'-end, the single-stranded region comprising a sequence capable of hybridizing to the oligonucleotide to be removed. The oligonucleotide may be comprised of RNA or DNA. In some embodiments, the single-stranded region comprises two regions of complementarity to the oligonucleotide to be removed adjacent to a single intermediate region. The intermediate region may be a non-nucleotide spacer or may be comprised of inosine nucleotides.

[0011] In some embodiments, the present invention is a method of detecting multiple targets in multiple cells in a reaction mixture, comprising: binding multiple unique binders to the targets in the multiple cells, each specific to one of the targets; adding multiple subcode oligonucleotides to each of the binders in the multiple cells in an ordered manner during successive rounds of split pool synthesis, where the subcode oligonucleotides in each round anneal adjacently to the subcode oligonucleotides from the previous round via an annealing region; covalently linking the adjacently annealed subcode oligonucleotides to each other to create a unique cell-origin nucleotide code in each cell; and removing excess subcode oligonucleotides 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'-end and the 3'-end that can hybridize to the subcode oligonucleotide. The method may further comprise contacting the sample with 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 equal in length to the barcode region flanked by two sequences that can hybridize to the annealing regions in the subcode oligonucleotide. The spacer may be a carbon linker or may comprise an inosine-containing nucleotide.

[0012] In some embodiments, the present invention is a method for preparing a solution of amplified target nucleic acid that does not contain excess amplification primers, 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 that supports nucleic acid synthesis; subjecting the reaction mixture to a thermal cycling profile suitable for annealing and extending the forward and reverse amplification primers; and after completion of the thermal cycling profile, contacting the reaction mixture with a nucleic acid ligase and a double hairpin nucleic acid consisting 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'-end and the 3'-end that can hybridize to the forward or reverse amplification primer; ligating the forward and reverse amplification primers to the corresponding double hairpin nucleic acid, thereby removing the forward and reverse amplification primers from the solution of amplified target nucleic acid. The method may further comprise contacting the sample with polynucleotide kinase.

[0013] In some embodiments, the present invention is a method for preparing a solution of amplified target nucleic acid that does not contain excess amplification primers, comprising contacting a reaction mixture containing the target nucleic acid with a thermostable ligase in the presence of a first probe and a second probe that can hybridize adjacent to the target nucleic acid and a reagent that supports ligation, subjecting the reaction mixture to a thermal cycling profile suitable for annealing and ligating the first and second probes to each other, and after completion of the thermal cycling profile, contacting the reaction mixture with a double hairpin nucleic acid consisting 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'-end and the 3'-end that can hybridize to the first probe or the second probe, and 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 comprise contacting the sample with polynucleotide kinase.

[0014] In some embodiments, the present invention is a method for forming a library of nucleic acids that does not contain excess adapter molecules, comprising contacting a reaction mixture containing nucleic acids with an adapter molecule and a ligase, ligating the adapter molecule to an end of the nucleic acid, contacting the reaction mixture with a double hairpin nucleic acid consisting 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'-end and the 3'-end that can hybridize to the adapter molecule, and ligating the adapter molecule to the double hairpin nucleic acid, thereby removing the adapter molecule from a solution of amplified target nucleic acid. In some embodiments, the adapter molecule consists of two oligonucleotides that form at least one double-stranded region. In some embodiments, the reaction mixture is subjected to a high temperature sufficient to separate the adapter molecules 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 adapter molecule. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram of a double hairpin structure and method of use. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention The term "nucleic acid" refers to a polymer of nucleotides and includes, unless otherwise limited, known analogues of natural nucleotides that can function (eg, hybridize) in a manner similar to naturally occurring nucleotides.

[0017] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, intergenic DNA, loci defined from linkage 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), a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA); DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe molecules, and amplification primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to nucleotide structure, if present, may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. When polynucleotide sequences are provided, they are listed in the 5' to 3' direction unless otherwise indicated. The term "oligonucleotide" refers to shorter nucleic acids, typically 100 nucleotides or less in length, although longer nucleic acids may also be referred to as oligonucleotides.

[0018] A nucleic acid "probe" is an oligonucleotide that can bind to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, and thus form a double-stranded structure. The probe binds or hybridizes to a "probe binding site". The probe can be labeled with a detectable label to allow easy detection of the probe, especially after the probe hybridizes to its complementary target. However, alternatively, the probe can be unlabeled but can be directly or indirectly detectable by specific binding with a labeled ligand.

[0019] The terms "epitope" and "target molecule" are used interchangeably herein to refer to a molecule of interest (protein or nucleic acid) that is detected and / or quantified by the methods described herein.

[0020] The term "targeted oligonucleotide" refers to the oligonucleotide to be removed or depleted from the reaction mixture by the novel method disclosed herein. Although the targeted oligonucleotide may share similarities (e.g., share some nucleic acid sequences) with the target molecule defined above, targeting the targeted oligonucleotide only refers to removing the targeted oligonucleotide from the reaction mixture according to the method disclosed herein. The targeted oligonucleotide is interchangeably referred to as "excess oligonucleotide", "undesired oligonucleotide", "oligonucleotide to be depleted" and "oligonucleotide to be removed".

[0021] The term "hairpin" in reference to nucleic acids refers to a secondary structure formed by single strands of nucleic acid that have at least portions that are complementary to each other and can anneal to each other. A hairpin contains a short intervening region between the two complementary regions such that the nucleic acid strands make a sharp bend upon annealing. Hairpins with longer intervening regions do not form a sharp bend, but instead form a loop, and this structure is sometimes called a "stem-loop" structure. There is no precise delineation between hairpins and stem-loops. Stem-loop structures with smaller loops are sometimes called hairpins.

[0022] The present invention is an improved method or removal of excess or undesired oligonucleotides from a reaction mixture without a purification step, which allows for easy removal or inactivation of oligonucleotides with little or no implementation time and without loss of the desired product.

[0023] In reactions involving a mixture of oligonucleotides, it is important to control and remove by-products and excess reagents, as they can serve as substrates for problematic side reactions.Side reactions, such as primer-primer or probe-probe interactions, off-target priming and off-target probe binding, affect the kinetics of intended reactions by sequestering reactants or consuming reaction components.The invention disclosed herein includes methods and compositions that make specific oligonucleotide sequences inert to side reactions and off-target base pairing.

[0024] In some embodiments, the invention includes a short oligonucleotide (DNA or RNA) referred to herein as a "double hairpin" (FIG. 1). After completion of a reaction including an oligonucleotide that removes unused excess, the double hairpin is added to the reaction mixture. The sequence of this double hairpin oligonucleotide is designed such that it 1) forms a hairpin at each of its ends and 2) has a middle section between the hairpins that binds to the oligonucleotide to be removed or rendered inactive (the "target oligonucleotide"). When the double hairpin oligonucleotide is added to a mixture containing the target oligonucleotide, the target oligonucleotide anneals to the middle section of the double hairpin. Upon annealing, a ligase enzyme is used to form covalent bonds between both the 5'- and 3'-ends of the double hairpin oligonucleotide and both the 5'- and 3'-ends of the target oligonucleotide, thus forming a topologically circular, inactive product.

[0025] The circularization product renders the target oligonucleotide inactive in several ways. First, ligation prevents the 3'-end of the target oligonucleotide from serving as an amplification primer in any primer extension reaction, including exponential amplification reactions. Second, base pairing with the double hairpin oligonucleotide prevents the target oligonucleotide from base pairing with other nucleic acids in the reaction mixture.

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

[0027] In some embodiments, nucleic acid needs to be isolated from the sample. Methods of DNA extraction are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd Edition, Cold Spring Harbor Laboratory Press, New York, NY). A variety of kits are commercially available for extracting nucleic acid (DNA or RNA) from biological samples (e.g., BD Biosciences Clontech (Palo Alto, CA), Epicentre Technologies (Madison, WI), Gentra Systems, INC. (Minneapolis, MN), and Qiagen, INC. (Valencia, CA), Ambion, Inc. (Austin, TX), BioRad Laboratories (Hercules, CA), etc.

[0028] In some embodiments, purification is by affinity binding. In a variation of this embodiment, the affinity is for a specific target sequence (sequence capture). In other embodiments, the amplification primer comprises an affinity tag. Any affinity tag known in the art can be used, such as biotin, an antibody, or an antigen for which a specific antibody exists. The affinity partner for the affinity tag can be in solution, for example on a solution-phase solid support such as a suspended particle or bead, or can be bound to a solid-phase support.

[0029] In some embodiments, the nucleic acids are separated by size and purified using size exclusion chromatography, electrophoresis, or epitachophoresis.

[0030] A nucleic acid, protein or other marker of interest may be present in a cell or reaction mixture and may be the target of a detection or quantification procedure that is improved as disclosed herein by removal of 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 comprises a locus of a gene variant, such as a polymorphism, including a single nucleotide polymorphism or variant (SNP of SNV), or a gene rearrangement, such as resulting in a gene fusion. In some embodiments, the protein biomarker comprises an amino acid change that results in the creation of a unique epitope. In some embodiments, the target nucleic acid or target protein comprises a biomarker, i.e., a gene or protein antigen whose variants are associated with a disease or condition. For example, the target nucleic acid and protein are described in U.S. Patent Application Serial No. 14 / 774,518, filed September 10, 2015. Such a panel is available as the AVENIO ctDNA Analysis Kit (Roche Sequencing Solutions, Pleasanton, Calif.). In other embodiments, the target nucleic acid and protein are characteristic of a particular organism, aiding in the identification of the organism or characteristics of the pathogenic organism, such as drug susceptibility or drug resistance. In yet other embodiments, the target nucleic acid or protein is a combination of HLA or KIR sequences that define a unique characteristic of a human subject, such as a unique HLA or KIR genotype of the subject. In yet other embodiments, the target nucleic acid is a somatic sequence, such as a rearranged immune sequence representing an immunoglobulin (including IgG, IgM and IgA immunoglobulins) or a T cell receptor sequence (TCR). In yet another application, the target is a fetal sequence present in maternal blood, including fetal sequences characteristic of a fetal disease or condition or a maternal condition associated with pregnancy. For example, the target can be one or more of the autosomal or X-linked disorders described in 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 cells. 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 (e.g., Pdcd1, Havrcr2, Lag3, CD244, Entpd1, CD38, CD101, Tigit, CTLA4), cell surface receptors (e.g., TNFRSF9, TNFRSF4, Klrg1, CD28, Icos, IL2Rb, IL7R) or chemokine receptors (e.g., 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] With reference to Figure 1, the present invention includes a novel composition comprising a double hairpin oligonucleotide, also referred to as a double hairpin nucleic acid, for capturing undesired oligonucleotides from a reaction mixture. The oligonucleotide comprises three portions: a first hairpin at the 5'-end, a second hairpin at the 3'-end, and a single-stranded region between the 5'-end and the 3'-end hairpin, which can hybridize to an oligonucleotide to be removed ("target oligonucleotide").

[0035] Each hairpin is formed by two sequences within the oligonucleotide that can form a stable hybrid with each other and form a DNA (or RNA) bend between the hybridized sequences. In some embodiments, one or both hairpins are formed by regions that are completely complementary to each other. In other embodiments, one or both hairpins are formed by only partially complementary sequences, and still form a stable hairpin.

[0036] In some embodiments, one or both of the hairpin regions of the double hairpin oligonucleotide contain one or more modified nucleotides that increase the thermal stability (melting temperature, Tm) of the hairpin structure. For example, one or both hairpin portions contain one or more of 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutyne-2'-deoxyuridine, 8-aza-7-deazaguanosine, ribonucleotides, 2'O-methylribonucleotides, or locked nucleic acids.

[0037] In some embodiments, the hairpin-forming sequence is an artificial, optionally in silico designed nucleic acid sequence. In other embodiments, the hairpin-forming sequence is a naturally occurring hairpin-forming sequence as reviewed in 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, so as to allow the target oligonucleotide to link with both ends of the double hairpin oligonucleotide. Those skilled in the art will understand that a stable hybrid can be formed by nucleic acid strands that are not completely complementary to each other, as well as completely complementary to each other. 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 completely complementary to the target oligonucleotide. In some embodiments, a part of the single-stranded region of the double hairpin oligonucleotide is completely complementary to the target oligonucleotide. In some embodiments, the 5'-end adjacent portion and the 3'-end adjacent portion of the single-stranded region are completely complementary to the 3'-end adjacent portion and the 5'-end adjacent portion of the target oligonucleotide, but the middle portion of the single-stranded region is not complementary to the middle portion of the target oligonucleotide, or is only partially complementary to it.

[0039] In some embodiments, the unfolded double hairpin oligonucleotide is about 40 to about 200 nucleotides long. The hairpin-forming sequence is about 9 to about 40 nucleotides long. There is no prescribed length for either of the two hairpin regions, but it is desirable to have a region of sufficient length to ensure the stability of the hairpin in the reaction mixture. Those skilled in the art have access to a variety of tools that predict the stability of nucleic acid duplexes (melting temperature, Tm) under various temperature and salt conditions. The single-stranded region is custom-made to accommodate the target oligonucleotide. Oligonucleotides, including amplification primers, probes and adapter molecules, typically range from 10 to 100 nucleotides long, with the majority being between 20 and 50 nucleotides long. Thus, the single-stranded region of the double hairpin oligonucleotide ranges from about 10 to about 100 nucleotides long, most frequently 20 to 50 nucleotides long.

[0040] In some embodiments, the present invention is a method for removing excess or undesired oligonucleotides ("target oligonucleotides") from a reaction mixture. The method includes contacting a reaction mixture containing the target oligonucleotide with a double hairpin nucleic acid that includes 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'-end and the 3'-end. The single-stranded region includes a sequence that can hybridize to the target oligonucleotide.

[0041] The double hairpin oligonucleotide is added at a concentration calculated to exceed the concentration of the target oligonucleotide to be removed, so as to ensure good 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 other cases, the optimal molar concentration of the double hairpin oligonucleotide is experimentally determined by titrating the double hairpin oligonucleotide.One distinct advantage of the present invention is that the double hairpin oligonucleotide is designed to interact with the target oligonucleotide and not interact with other nucleic acids in the reaction mixture.

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

[0043] In some embodiments, the reaction is incubated for the time required for the formation of a hybrid between the target oligonucleotide and the double hairpin oligonucleotide, which in some embodiments is between 5 and 15 minutes.

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

[0045] After the target oligonucleotide has annealed to the single-stranded region of the double hairpin oligonucleotide, the reaction mixture is contacted with a ligase. Ligase enzymes are present to ligate both DNA and RNA, and accommodate both DNA and RNA double hairpin oligonucleotides and target oligonucleotides. A ligase appropriate for the type of nucleic acid in 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 cofactors and substrates (e.g., ATP) are already present in the reaction mixture due to upstream processes. In such embodiments, no additional ligase or ligase cofactors are added to remove the target oligonucleotide. In other embodiments, ligase and ligase cofactors 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 polynucleotide kinase (PNK) and any necessary cofactors, such as ATP, to phosphorylate the 5'-end and allow ligation to occur.

[0048] In some embodiments, enzymes (e.g., ligases and polynucleotide kinases) are removed or inactivated (e.g., heat inactivated) prior to downstream processing involving reaction mixtures depleted of target oligonucleotides.

[0049] Those skilled in the art will understand that the term "removal" in relation to a target oligonucleotide includes all forms of inactivation of the target oligonucleotide. When the target oligonucleotide can no longer hybridize to any nucleic acid in the reaction mixture and prime nucleic acid synthesis, the oligonucleotide is effectively removed from the reaction mixture. Thus, the sequestration of the target oligonucleotide within the double-stranded covalently closed topologically circular nucleic acid formed by the double hairpin oligonucleotide and the target oligonucleotide (Figure 1 bottom) constitutes the removal of the target oligonucleotide from the reaction mixture.

[0050] In some embodiments, the present invention is an improved method of performing nucleic acid amplification via polymerase chain reaction (PCR). As described in detail in US Pat. No. 4,683,195, polymerase chain reaction (PCR) involves contacting a sample solution containing nucleic acids with a forward amplification primer, a reverse amplification primer, a nucleic acid polymerase, preferably a thermostable polymerase, and nucleic acid precursors, such as dNTPs or dNTP analogs, that can be incorporated by the nucleic acid polymerase. The reaction mixture is subjected to a thermal cycling profile that includes multiple cycles including a DNA denaturation step (90° C. or higher), an optional primer annealing step (45-72° C.) and a polymerase extension step (65-72° C.). In some embodiments, the PCR is a real-time PCR that includes fluorescently labeled detection probes, for example as described in US Pat. No. 5,804,375. In state of the art protocols, after the amplification reaction is completed, the reaction mixture is subjected to a purification step, such as a SPRI bead cleanup, to remove excess amplification primers. The present invention dispenses with the purification step.

[0051] In some embodiments, after the amplification reaction is completed, the reaction mixture is contacted with the novel double hairpin oligonucleotide described herein. In some embodiments, the target oligonucleotide is a mixture of forward and reverse amplification primers. In some embodiments, the target oligonucleotide mixture also includes a detection probe. The double hairpin oligonucleotide is a combination of two or three oligonucleotides. The first double hairpin oligonucleotide includes a middle region that is complementary to the forward amplification primer. The second double hairpin oligonucleotide includes a middle region that is complementary to the reverse amplification primer. There may also be a third double hairpin oligonucleotide that targets the detection probe. The third double hairpin oligonucleotide includes a middle region that is complementary to the detection probe. The excess amplification primer and, if present, the probe are ligated to the corresponding double hairpin oligonucleotide to form a closed circular structure.

[0052] In some embodiments, prior to ligation, the reaction mixture is contacted with polynucleotide kinase (PNK) and any necessary cofactors and substrates, such as ATP, to phosphorylate the 5'-ends of the forward and reverse amplification primers and the probe, if present, to allow ligation to occur.

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

[0054] After the excess amplification primer and, if present, excess probe, are sequestered in a closed circular structure, downstream processing can proceed immediately.

[0055] In some embodiments, the present invention is an improved method of performing nucleic acid amplification via ligase chain reaction (LCR). As described in detail in US Pat. No. 6,312,892, ligase chain reaction (LCR) involves contacting a sample solution containing nucleic acid with a probe set including a first oligonucleotide probe and a second oligonucleotide probe capable of adjacently hybridizing to a nucleic acid template, and a ligase, preferably a thermostable ligase. The reaction mixture is subjected to a thermal cycling profile including multiple cycles including a DNA denaturation step (90°C or higher), an optional probe annealing step (45-65°C) and a ligation step (65°C or lower). In state of the art protocols, after the amplification reaction is completed, the reaction mixture is subjected to a purification step, such as a SPRI bead cleanup, to remove excess probe. The present invention dispenses with the purification step.

[0056] In some embodiments, after the completion of the amplification reaction, 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 comprises a middle region that is complementary to the first probe. The second double hairpin oligonucleotide comprises a middle region that is complementary to the second probe. Advantageously, the ligase and all necessary cofactors are already present in the reaction mixture. The excess probe is ligated to the corresponding double hairpin oligonucleotide to form a closed circular structure.

[0057] In some embodiments, prior to ligation, the reaction mixture is contacted with polynucleotide kinase (PNK) and any necessary cofactors and substrates, such as ATP, to phosphorylate the 5'-ends of the probes to allow ligation to occur.

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

[0059] Excess amplification primers and, if present, probes are sequestered into closed circular structures after which downstream processing can proceed immediately.

[0060] In some embodiments, the present invention is an improved method of performing quantum barcoding (QBC) to detect multiple targets in multiple individual cells (US Pat. No. 10,144,950). The method includes preparing a cell suspension. The cells are then contacted with a unique binding agent, such as a DNA or RNA probe (including aptamers) or an antibody. The probe or antibody includes at least one moiety or element that specifically interacts with the target and an element that allows for the construction of a combinatorial nucleic acid barcode. In some embodiments, the assay is a multiplex assay, whereby multiple target molecules are detected in multiple cells in a single reaction mixture using multiple different binding agents of the same or different types (e.g., multiple different nucleic acid probes, or multiple different antibodies, or a combination of nucleic acid probes and antibodies). In the case of a unique binding agent in an antibody, the antibody may include a linker oligonucleotide that facilitates the construction of the barcode. Methods for attaching nucleic acids to antibodies are known, for example, Gullberg et al., PNAS 101(22): pages 228420-8424 (2004); Boozer et al., Analytical Chemistry, 76(23): pages 6967-6972 (2004) or Kozlov et al., Biopolymers 5:73(5): pages 621-630 (2004). When the unique binding agent is a nucleic acid probe, the barcode can be directly attached to the probe.

[0061] After the unique binders bind to their targets, the probe or antibody-bound cells are subjected to split-pool barcode assembly, which is described in more detail in U.S. Pat. No. 10,144,950, incorporated herein by reference. A unique cell origin code is constructed on each cell with a unique binder bound. The unique cell barcode is a modular structure assembled from subunits by stepwise addition of the subunits. Each subunit contains a barcode and an attachment region that serves to attach the subunit to the growing unique cell barcode. The subunits are attached to each other or to a common scaffold via attachment regions, e.g., complementary nucleic acid sequences. Attachment may include one or both of hybridization to the scaffold or adjacent subunits and linkage to adjacent subunits. After the unique cell barcodes are 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 present invention, the reaction mixture containing the unique barcode is contacted with the novel double hairpin oligonucleotide described herein prior to the amplification step. The target oligonucleotide is a mixture of multiple barcode subunits. The double hairpin oligonucleotide is designed to bind to one or more, including all barcode subunits in the mixture. In some embodiments, the single-stranded portion of the double hairpin oligonucleotide comprises two regions capable of hybridizing to the annealing regions of the barcode subunits, and is adjacent to a single intermediate region having the length of the subcode contained in the barcode subunit. Since the subcodes differ between 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 an inosine-containing nucleic acid having a non-nucleotide spacer of a length corresponding to the length of the subcode or a number of inosine nucleotides corresponding to the number of nucleotides in the subcode.

[0063] In some embodiments, the barcode subunit oligonucleotides can be ligated to a unique barcode, i.e., already advantageously contain a 5'-phosphate. Moreover, in such embodiments, an active ligase is already advantageously present in the reaction mixture. In such embodiments, the reaction mixture is contacted only with the double hairpin oligonucleotide.

[0064] In other embodiments, the barcode subunits are added to the unique barcode by a method other than ligation (see, e.g., embodiments in which the barcode is copied by a polymerase, see U.S. Patent Application No. 16 / 250,974, filed January 19, 2019). In such embodiments, the reaction mixture is contacted with a double hairpin oligonucleotide and further contacted with a ligase. If the barcode subunit lacks a 5'-phosphate, the reaction mixture is further contacted with polynucleotide kinase (PNK). In some embodiments, the reaction mixture is contacted with ATP, a necessary cofactor for the kinase and ligase.

[0065] The excess barcode subunits are ligated to the double hairpin oligonucleotide to form a closed circular structure. Following sequestration of the excess barcode subunit oligonucleotides in the closed circular structure, amplification can proceed immediately. The barcode oligonucleotides can no longer interfere with nucleic acid amplification, for example by acting as amplification primers.

[0066] It is particularly advantageous that the present invention does not require a purification step prior to amplification. Quantum barcoding (QBC) is a method of single-cell analysis in which each unique cell barcode represents a single cell. Thus, the loss of a single unique cell barcode molecule represents the loss of a valuable data point. In the methods described herein, downstream analysis steps can proceed immediately after removal of excess barcode subunits.

[0067] In some embodiments, the present invention is an improved method of forming libraries for nucleic acid sequencing, in which nucleic acids in a sample are ligated to adapter molecules and excess adapter molecules are removed prior to downstream processing of the library.

[0068] In some embodiments, the method includes a step of treating the nucleic acid in the sample before ligation of the adaptor molecule. The sample nucleic acid may already be blunt-ended or may be blunt-ended by enzymatic treatment (e.g., "end repair"). In other embodiments, the blunt-ended DNA undergoes A-tailing, in which a single A nucleotide is added to the 3'-end of one or both blunt ends. The adaptor molecule may be a short nucleic acid that is double-stranded or partially double-stranded, with one blunt end having a single T nucleotide extending from the blunt end to facilitate ligation between the sample nucleic acid and the adaptor molecule. Commercially available kits for performing end repair, A-tailing and adaptor ligation include AVENIO ctDNA Library Prep Kit or KAPA HyperPrep and HyperPlus kits (Roche Sequencing Solutions, Pleasanton, Calif.).

[0069] In some embodiments, the present invention includes a novel step of removing excess adapter molecules from a library formation reaction by sequestering them in a closed circular structure formed by ligation to the double hairpin oligonucleotide described herein. The target oligonucleotide comprises both strands of the adapter molecule. The double hairpin is a combination of two double hairpin oligonucleotides. The first double hairpin oligonucleotide comprises a middle region that is complementary to the first strand of the adapter molecule, and the second double hairpin oligonucleotide comprises a middle region that is complementary to the second strand of the adapter molecule.

[0070] In some embodiments, the method further comprises incubating the reaction mixture containing the library and excess adapter molecules at a high enough temperature to separate the strands of the adapter molecules but not the strands of the library nucleic acid or hairpin structure in the double hairpin oligonucleotide.To facilitate this step of the method, the double hairpin oligonucleotide may comprise modified nucleotides that increase the melting temperature (Tm) of the hairpin.For example, one or both hairpin portions comprise one or more of 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutyne-2'-deoxyuridine, 8-aza-7-deazaguanosine, ribonucleotides, 2'O-methylribonucleotides or locked nucleic acids.

[0071] In some embodiments, the reaction mixture is also contacted with a polynucleotide kinase to phosphorylate the 5'-end of the adapter molecule.

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

[0073] The excess oligonucleotide removal methods of the present invention are not limited to the exemplary applications specifically addressed above, but may be used in any diagnostic, prognostic, therapeutic, patient stratification, drug development, treatment selection and screening process involving the use of oligonucleotides, where it is desirable to remove excess oligonucleotides.

Claims

1. 1. A method for removing undesired oligonucleotides from a reaction mixture, comprising: a. subjecting the reaction mixture to 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, the single-stranded region comprising a sequence capable of hybridizing to the oligonucleotide to be removed; contacting a double hairpin nucleic acid comprising a single nucleic acid strand having the formula: b. annealing the oligonucleotide to be removed to the double hairpin nucleic acid; c. ligating the oligonucleotide to be removed to the end of the double hairpin nucleic acid to form a circular inactive product, thereby removing the undesired oligonucleotide from the reaction mixture; The method comprising:

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

3. The method of claim 1 or 2, wherein the reaction mixture comprises a ligase prior to step a.

4. The method of any one of claims 1 to 3, wherein the double hairpin nucleic acid comprises a 5'-phosphate group.

5. The method of any one of claims 1 to 4, wherein the single-stranded region comprises two regions of complementarity to the oligonucleotide to be removed, flanked by 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 an inosine nucleotide.

8. 6. The method of claim 5, wherein the oligonucleotides to be removed comprise a plurality of oligonucleotides having two constant regions flanking a single barcode region that varies among the plurality of oligonucleotides.

9. 1. A method for detecting multiple targets in multiple cells in a reaction mixture, comprising: a. binding to said targets in a plurality of cells with a plurality of unique binding agents, each binding agent being specific for one of said targets; b. adding a plurality of subcode oligonucleotides in an ordered manner to each of the binding agents in the plurality of cells during successive rounds of split pool synthesis, wherein the subcode oligonucleotides in each round anneal adjacently to the subcode oligonucleotides from the previous round via annealing regions, and covalently linking the adjacently annealed subcode oligonucleotides to each other to create a unique cell-origin nucleotide code in each cell; c) 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'-end and the 3'-end capable of hybridizing to the subcode oligonucleotide, and ligating excess subcode oligonucleotides of the corresponding double hairpin nucleic acid to form a circular inactive product, thereby removing excess subcode oligonucleotides; The method comprising:

10. 10. The method of claim 9, wherein 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 equal in length to the barcode region flanked by two sequences capable of hybridizing to the annealing regions in the subcode oligonucleotide.

11. 1. A method for preparing a solution of amplified target nucleic acid that is free of excess amplification primers, comprising: a. contacting a reaction mixture containing a target nucleic acid with forward and reverse amplification primers and a thermostable nucleic acid polymerase in the presence of reagents that support nucleic acid synthesis; b. subjecting the reaction mixture to a thermal cycling profile suitable for annealing and extension of the forward and reverse amplification primers; c. After completion of the thermal cycling profile, the reaction mixture is reacted with a nucleic acid ligase and 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, capable of hybridizing to the forward amplification primer or the reverse amplification primer; contacting a double hairpin nucleic acid with a single nucleic acid strand having d. ligating the forward amplification primer and the reverse amplification primer to the corresponding double hairpin nucleic acid to form a circular inactive product, thereby removing the forward amplification primer and the reverse amplification primer from the solution of amplified target nucleic acid; The method comprising:

12. 1. A method for preparing a solution of amplified target nucleic acid that is free of excess probe, comprising the steps of: a. contacting a reaction mixture containing a target nucleic acid with a thermostable ligase in the presence of a first probe and a second probe capable of adjacently hybridizing to said target nucleic acid, and a reagent that supports ligation; b. subjecting the reaction mixture to a thermal cycling profile suitable for annealing and ligating the first probe and the second probe to one another; c. After completion of the thermal cycling profile, 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, capable of hybridizing to the first probe or the second probe; contacting a double hairpin nucleic acid with a single nucleic acid strand having d. ligating the first probe and the second probe to the corresponding double hairpin nucleic acid to form a circular inactive product, thereby removing the first probe and the second probe from the solution of amplified target nucleic acid; The method comprising:

13. 1. A method for forming a library of nucleic acids that does not contain excess adaptor molecules, comprising: a. contacting a reaction mixture comprising nucleic acids with an adapter molecule and a ligase; b. ligating the adapter molecule to the end of the nucleic acid; c. The reaction mixture is 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, the single-stranded region being capable of hybridizing to the adapter molecule; contacting a double hairpin nucleic acid with a single nucleic acid strand having d. ligating the adapter molecule to the double hairpin nucleic acid to form a circular inactive product, thereby removing the adapter molecule from the solution of amplified target nucleic acid; The method comprising:

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