# Patent

## Title
DNA glycosylase/lyase and AP endonuclease substrates

## Abstract

A new class of nucleic acid substrates for AP endonucleases and members of the glycosylase/lyase family of enzymes is described. Representatives of each family, the enzymes Nfo and fpg, respectively, cleave nucleic acid backbones at positions in which a base has been replaced by a linker to which a variety of label moieties may be attached. The use of these synthetic substrates embedded within oligonucleotides is of utility in a number of applications.

## Background
RELATED APPLICATIONS
This application is a continuation and claims the benefit of priority to U.S. patent application Ser. No. 12/800,633, filed May 18, 2010, which claims the benefit of priority to U.S. Provisional Patent Application No. 61/179,793 filed May 20, 2009, which is hereby incorporated by reference in its entirety.


FIELD OF THE INVENTION
This invention relates to oligonucleotide substrates, as well as for their uses, for example, as probes for nucleic acid amplification reactions.
BACKGROUND OF THE INVENTION
Enzymes which metabolize nucleic acids in a manner specified by primary sequence, backbone structure, or a base character (often damaged or modified base) are of utility in biotechnology applications. Several families of such enzymes are used routinely in nucleic acid-based techniques and include restriction endonucleases, polymerases, ligases and exonucleases. Additionally, a variety of single-subunit (non-restriction) endonucleases which rely not on specific sequence strings but on recognising unusual, damaged or missing bases have been described over the years. These enzymes can be loosely divided into 2 groups—the AP endonucleases, of which E. coli endonuclease IV (Nfo) and E. coli exonuclease III are examples, and the DNA glycosylase/lyase family of which E. coli fpg, MUG and Nth are examples.
The AP endonucleases are characterised by the ability to recognise and cleave the sugar-phosphate backbone at abasic sites (other enzymatic activities may also be present) when found in the context of duplex DNA. Recognition and incision at abasic sites occurs in a biochemical manner that is distinct to the glycosylase/lyase family and not by beta-elimination or beta/delta-elimination. Consequently they attack not only true abasic sites but other substrates including tetrayhydrofuran moieties which lack an oxygen atom on the 1′ carbon of the sugar ring (Takeshita et al., 1987, J Biol Chem. 262(21):10171)(see FIG. 1 for chemical structures).
In contrast, glycosylase/lyase enzymes including the fpg protein (8-oxoguanine DNA glycosylase, fpg in E. coli and OGG1 in mammals) or Nth proteins (endonuclease III in E. coli, Nth1 in humans, etc.) function in a 2-stage catalytic manner in which damaged bases are first recognized and excised via formation of a Schiff base between the protein and the DNA, and secondly the abasic site thus generated is processed by beta-elimination or beta-delta elimination in a manner distinct to the AP endonucleases. In this case tetrahydrofuran (THF) residues are not a substrate for lyase activity as no C1′ oxygen atom is present in this abasic mimic and such sugars lacking oxygen at the 1′ position are resistant to attack (Takeshita et al., 1987)(FIG. 1).
The use of AP endonucleases and glycosylase/lyases in molecular biology techniques has been described. One application is the use of these enzymes to process substrates generated during in vitro DNA amplification reactions, or similar kinds of applications, and in particular when a synthetic ‘probe’ oligonucleotide has been provided containing modified sugars or bases which can become a substrate for the enzymes if the synthetic oligonucleotide hybridizes specifically to molecules in the sample. An example of such an application is given in U.S. Pat. No. 7,435,561 B2 and Piepenburg et al., Plos Biology, 2006 4(7):e204 in which tetrahydrofuran-modified oligonucleotides are used as substrates for the E. coli Nfo (endonuclease IV) protein as a method to measure DNA amplification (Nfo is one of the two AP endonucleases of E. coli).
Application of glycosylase/lyases to similar strategies can also be envisioned. The ability of fpg protein to similarly process modified bases such as 8-oxoguanine within a DNA amplification reaction for the purposes of reaction-monitoring has been described (U.S. Pat. No. 7,435,561 B2). Furthermore the fact that glycosylase/lyase enzymes such as fpg and Nth do not leave 3′ extendable ends but rather blocked 3′ ends (due to the differences in catalytic mode) may have particular utility in circumstances in which one wishes to ensure that the processed probe cannot be a ready substrate for polymerases or other activities dependent on a 3′ hydroxyl moiety.
Despite the potential of these enzymes, they possess certain features that make them unattractive for use in some applications. Notably, unlike the THF residue, true abasic sites required for the backbone-incising activity of DNA lyases are not stable under physiological conditions and are quickly hydrolyzed in aqueous solutions making them impractical for use in most molecular procedures. Instead specific damaged bases can be incorporated and used as the primary substrates for the glycosylase activity to generate the abasic site transiently before backbone hydrolysis by the lyase activity. Unfortunately however, typical damaged base analogs such as 8-oxoguanine (fpg) or thymidine glycol (Nth) tend to be rather expensive to synthesize and also impart sequence requirements on the probe as ideally they must be paired opposite specific bases on the opposing strand. In principle it would be far more convenient to have a stable substrate analogous to the generic THF residue that can be employed for AP endonucleases but retaining reactivity with the lyase activity of glycosylase/lyase enzymes.
Here we show that the fpg protein, as well as the AP endonuclease IV of E. coli (Nfo), efficiently cleaves DNA backbones containing a variety of substrates that lack a base but contain a 1′-oxygen atom covalently attached to a carbon-based linker [C]n. The linker can itself be used to attach other moieties such as biotin, fluorophores and other coupled groups, particularly useful if an amine-ended linker can be used to couple a variety of agents. Surprisingly, nucleotides having this arrangement and referred to generally as dR-O—[C]n appear to be good substrates of the fpg protein in a number of contexts, and are also substrates for the endonuclease IV protein, but appear relatively poor substrates for E. coli exonuclease III. We anticipate the use of oligonucleotides containing such dR-O—[C]n groups as substrates in a number of circumstances, in particular within in vitro reactions such as part of detection strategies for nucleic acid detection methods. The length of the linker used in this study is 6 carbon atoms, as available on certain commercially available nucleotides, however it is anticipated that a variety of carbon chain lengths might be employed and that it is the carbon-oxygen-carbon structure with little subsequent steric bulking that affords these structures sufficient plasticity to the enzymes.
SUMMARY OF THE INVENTION
The present invention relates in part to the discovery that AP endonucleases, DNA glycosylases, an DNA glycosylase/lyases, such as fpg and Nfo proteins, can catalyze the breaking of the DNA backbone at sites containing dR-O—[C]n residues in which no base is present at the C1′ position of the sugar, but that retains an oxygen atom at that position. The oxygen atom bridges the sugar to a carbon atom of a carbon linker with n (e.g., 1-8) carbon atoms (i.e., [C]n). Consequently nucleic acid probes can be constructed containing dR-O—[C]n residues by the use of commercially available phosphoramidites and can be substrates for AP endonucleases and DNA glycosylase/lyase enzymes if they form duplexes with complementary nucleic acids. A variety of moieties may be coupled to the linker portion of the dR-O—[C]n including fluorophores and other labels suggesting a number of strategies to detect successful processing of the probe as evidence of presence of a specific target nucleic acid. Applicants show how probes may be constructed using fluorescent molecules and quenchers using dR-O—[C]n as targeting sites for fpg, Nfo or other potential AP endonucleases or lyases. Applicants contemplate other uses of the dR-O—[C]n substrates in other detection schemes. For example, the dR-O—[C]n residue may be conjugated to a detactable label, where the activity of the nuclease frees the label, which can then be detected either immediately or via a subsequent process, via a measurable difference between the conjugated and free state.
In one aspect, processes are provided herein for cleaving an oligonucleotide containing a dR-O—[C]n residue that forms a duplex with a nucleic acid, by contacting the duplex with a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases. In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot).
In some embodiments, the process further comprises the step of contacting the oligonucleotide with the nucleic acid to form the oligonucleotide/nucleic acid duplex. In some embodiments, this comprises hybridizing the oligonucleotide to the nucleic acid. In some embodiments, this comprises (i) contacting the oligonucleotide with a recombinase to form a recombinase/oligonucleotide complex; and (ii) contacting the recombinase/oligonucleotide complex to the nucleic acid to form the oligonucleotide/nucleic acid duplex. In some embodiments, the nucleic acid is the product of a nucleic acid amplification reaction (e.g., a recombinase polymerase amplification (RPA) process or a polymerase chain reaction (PCR)).
In some embodiments, the process further comprises the step of detecting cleavage of the oligonucleotide. In some embodiments, the detection is monitored in real time. In some embodiments, the detection is monitored at an endpoint for the reaction.
In some embodiments, the oligonucleotide contains a fluorophore and a quencher, where one of the fluorophore or the quencher is conjugated to the carbon linker. The nuclease activity excises the conjugated fluorophore or quencher from the oligonucleotide and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
In another aspect, processes are provided herein for detecting the presence or absence of a target nucleic acid. The processes comprise the following steps: (a) contacting an oligonucleotide probe containing a dR-O—[C]n residue or nucleotide with the target nucleic acid to form a probe/nucleic acid duplex; (b) contacting the duplex with a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases to excise the linker from the complex and/or specifically cleave the probe at the dR-O—[C]n nucleotide; and (c) detecting whether such excision or cleavage has occurred. In some embodiments, the nucleic acid is the product of a nucleic acid amplification reaction (e.g., a recombinase polymerase amplification (RPA) process or a polymerase chain reaction (PCR)). In some embodiments, the amplification reaction is monitored in real time. In some embodiments, the amplification reaction is monitored at an endpoint for the reaction.
In some embodiments, the duplex is formed by hybridizing the probe to the nucleic acid. In some embodiments, the duplex is formed by (i) contacting the probe with a recombinase to form a recombinase/probe complex; and (ii) contacting the recombinase/probe complex to the nucleic acid to form the probe/nucleic acid duplex.
In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot).
In some embodiments, the probe contains a fluorophore and a quencher, where one of the fluorophore or the quencher is conjugated to the carbon linker. For example, the fluorophore and the quencher are separated by 4-6 bases in the probe. In some embodiments, the fluorophore or the quencher that is not conjugated to the carbon linker is conjugated to the end (e.g., the 5′-end) of the probe. The nuclease activity excises and frees the conjugated fluorophore or quencher associated with the dR-O—[C]n residue from the probe and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
In another aspect, provided herein are oligonucleotide probes containing a dR-O—[C]n residue. In some embodiments, the probes are 30 to 60 nucleotides in length and contain a fluorophore quencher pair separated by 10 nucleotides or less (e.g., 4-6 nucleotides), where either the flurophore or the quencher is conjugated to the dR-O—[C]n residue. In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the fluorophore or the quencher that is not conjugated to the dR-O—[C]n residue is conjugated to the end (e.g., the 5′-end) of the probe. In some embodiments, the probes are 30 to 40 nucleotides (e.g., 35 nucleotides) in length.
In yet another aspect, provided herein are kits comprising (i) an oligonucleotide containing a dR-O—[C]n residue, and (ii) a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases. In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg).
In yet a further aspect, provided herein are reaction mixtures comprising an oligonucleotide containing a dR-O—[C]n residue and a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases (e.g., endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg)). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot). In some embodiments, the reaction mixture is freeze dried or lyophilized.
In some embodiments, the reaction mixture further comprises a container. For example, the reaction mixture can be contained in a tube or in a well of a multi-well container. The reaction mixtures may be dried or attached onto a mobile solid support such as a bead or a strip, or a well.
In some embodiments, the reaction mixture further comprises a target or template nucleic acid that contains a sequence that is complementary to the oligonucleotide.
Other embodiments, objects, aspects, features, and advantages of the invention will be apparent from the accompanying description and claims. It is contemplated that whenever appropriate, any embodiment of the present invention can be combined with one or more other embodiments of the present invention, even though the embodiments are described under different aspects of the present invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Chemical structures: general structure of a normal abasic site containing a hydroxyl at the 1′ carbon position, of a tetrahydrofuran (THF) residue containing a hydrogen at the 1′ position, of the general dR-O—[C]n group indicating the position of the carbon-oxygen-carbon bridge between the C1′ of the DNA ribose group and the linker to the attached marker moiety, and finally of the dR-biotin nucleotide used in Example 1 and conforming to the dR-O—[C]n structure described.
FIG. 2 dR-biotin probe design: sequence (SEQ ID NO:3) and schematic representation of the oligonucleotide probe used to assess cleavage activity of Nfo and fpg proteins during RPA reactions in which a target sequence matching the probe sequence is amplified. The sequence of the oligonucleotide is indicated. The primer is labelled at the 5′ end with the FAM fluorophore, contains a dR-biotin within the body of the sequence, and is blocked by virtue of a 2′,3′dideoxycytidine residue.
FIG. 3 Comparison of amplification reactions lacking nuclease or containing Nfo or fpg enzyme: reveals that both enzymes can process the dR-biotin moiety giving rise to a faster migrating cleavage product and in the case of Nfo a product produced by extension of the cleavage product.
FIG. 4 Oligonucleotide probe design: example of a probe design, including an oligonucleotide body (here 35 nucleotides in length), a 5′-quencher modification (here a 5′-BHQ1), an internal dR-fluorophore nucleotide analogue in proximity to the quencher (here a dR-FAM at oligonucleotide position 6) and a 3′ polymerase extension block.
FIG. 5 Sensitivity and specificity: results of real-time fluorescence monitoring of two template titration experiments for the indicated human genomic targets using DNA dR-probes. In both cases the increase of fluorescence signal (relative to the baseline at 0 to 3 minutes) is only observed in reactions containing template and not in the no-template control. The onset time of the signal increase correlates with the amount of starting template (1000, 100 or 10 copies). Reaction time is in minutes (X-axis), fluorescence in arbitrary fluorescence units (Y-axis).
FIG. 6 Performance of different probes: results of real-time fluorescence monitoring of four sets of RPA reactions (in duplicates) for the indicated human genomic targets using DNA oligonucleotide probes of the design outlined in FIG. 2. The increase in fluorescence between 6 and 8 minutes results from the fpg-dependent processing of the dR-groups of the probes (here dR-FAM) and indicates ongoing DNA amplification and thus the presence of the target DNA template. Reaction time is in minutes (X-axis), fluorescence in arbitrary fluorescence units (Y-axis).



DETAILED DESCRIPTION OF THE INVENTION
The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present Specification will control.
The combination of enzymes with synthetic substrates for use in laboratory assays and manipulations is well known in the art. DNA repair endonucleases such as the glycosylases fpg and Nth, as well as AP endonucleases such as E. coli exonuclease III and endonuclease IV, are good examples of this combination—it is easy to generate synthetic substrates for these DNA repair enzymes by the use of modern oligonucleotide synthesis regimes and the wide existing variety of synthetic nucleotides that may be incorporated into DNA primers. These DNA repair enzymes can be readily employed for a variety of purposes, and one which has been recently exploited is their use as agents to assist the monitoring of isothermal Recombinase Polymerase Amplification (RPA) reactions.
RPA is a process in which recombinase-mediated targeting of oligonucleotides to DNA is coupled to DNA synthesis by a polymerase (U.S. Pat. No. 7,270,981 B2; U.S. Pat. No. 7,399,590; U.S. Pat. No. 7,435,561 B2; U.S. Pat. No. 7,485,428 B2; U.S. Pat. No. 7,666,598 B2 and foreign equivalents). RPA depends upon components of the cellular DNA replication and repair machinery, and relies upon establishment of a ‘dynamic’ recombination environment having adequate rates of both recombinase loading and unloading that maintains high levels of recombination activity achieved in the presence of specific crowding agents. RPA has the advantage that it combines the sensitivity, specificity and most other features of PCR but without the need for thermocycling and with extraordinary speed and robustness to off-temperature set-up. RPA has already benefited from the potential employment of a wide variety of nucleic acid processing enzymes such as known repair endonucleases which have been untapped by other processes because of either the need for thermostable equivalents or because they demonstrate poor regulation without accessory proteins such as single-stranded DNA binding proteins, a natural component of RPA reactions.
Briefly, RPA comprises the following steps: First, a recombinase agent is contacted with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer. Second, the first and second nucleoprotein primers are contacted to a double stranded target sequence to form a first double stranded structure at a first portion of said first strand and form a double stranded structure at a second portion of said second strand so the 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented towards each other on a given template DNA molecule. Third, the 3′ end of said first and second nucleoprotein primers are extended by DNA polymerases to generate first and second double stranded nucleic acids, and first and second displaced strands of nucleic acid. Finally, the second and third steps are repeated until a desired degree of amplification is reached.
Earlier work has demonstrated the extreme utility of the synthetic nucleotide tetrahydrofuran (THF) in the development of probe systems for the RPA method (Piepenburg et al., 2006; U.S. Pat. No. 7,435,561 B2). This base analog is oftentimes used to mimic abasic sites and has the natural advantage that it is stable—replacement of the 1′-hydroxyl of a natural abasic site with a hydrogen atom renders the nucleotides stable and unable to undergo spontaneous ring-opening and oligonucleotide fragmentation. This analog is readily available and cheap to incorporate into oligonucleotides. Due to differences in biochemical mechanism, however, while the E. coli AP endonucleases Nfo and ExoIII can cleave at such THF residues in synthetic primers, other DNA glycoslyase/lyases cannot. These latter enzymes normally require a damaged base (glycosylase activity) and/or the presence of a hydroxyl group at the 1′-position of the sugar (lyase activity) and THF is completely inert to their enzymatic activities. This presents something of a nuisance as these glycosylase/lyase enzymes could be useful tools also for in vitro reactions such as those in which a probe is processed in response to target DNA accumulation in RPA, or in other contexts and methods. More natural substrates, for example 8-oxoguanine for fpg, can be inserted into oligonucleotides to generate cleavage sites for these glycosylase/lyases, however these modifications are usually expensive, and furthermore often restrict the base which can be opposed to the modified nucleotide. Cheaper and more general nucleotide modifications which are substrates for these enzymes would be of great utility.
In an effort to explore the effects of a number of unusual base analogs as substrates for DNA repair enzymes we synthesised oligonucleotides containing nucleotides completely lacking a base, but retaining a carbon-oxygen-carbon linkage at the 1′ position of the sugar. Such nucleotide reagents are readily available and inexpensive, and are commonly used to incorporate labelling groups such as fluorophores or biotin into oligonucleotides within the body of the oligonucleotide. Commonly the carbon atom linked through oxygen to the 1′ carbon of the sugar is the first carbon atom of a linker which often ultimately ends with the labelling group, or alternatively an amine or other chemical moiety (e.g., a thiol) to which reagents may be readily coupled. Such reagents are often described in the literature as dR-X in which the dR refers to deoxyribose, and the X will often be linker-amine, or linker-fluorophore, or linker-biotin, or some other group or label. No-one has previously explored whether or not repair endonucleases would recognise such structures which lack a base but retain a carbon-oxygen-carbon covalent linkage at the 1′ sugar position. The absence of a hydroxyl means that the ring-opening processes of lyases should not operate without prior processing of the linker group and its associated excision. As known glycosylases normally operate on damaged bases rather than unusual carbon linkers there was no precedent to suggest that these dR-O—[C]n groups would be substrates for DNA glycosylase/lyases such as fpg.
FIG. 1 shows the general structure of a dR-O—[C]n group, as well as specifically the structure of the dR-biotin reagents as incorporated into oligonucleotides used herein and purchased from Eurogentec, Belgium. Such reagents used herein have a common 6 carbon atom linker between the 1′-sugar and a nitrogen atom which is often used to couple other reagents before or after oligonucleotide synthesis. In this study the biotin moiety of the dR-biotin oligonucleotide is linked via this nitrogen atom as an amide bond and then through a further 4 carbon atom linker. Other label reagents used in this study—dR-FAM and dR-Texas Red—are similarly arranged in which a fluorophore is coupled through an amide bond at the end of the 6 carbon atom linker.
A detectable label is defined as any moiety that may be detected using current methods. These labels include, at least, a fluorophore (also called a fluorescent molecule, fluorochrome), an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, a digoxygenin residue, a peptide, and a combination thereof.
“A member of a binding pair” is meant to be one of a first and a second moiety, wherein said first and said second moiety have a specific binding affinity for each other. Suitable binding pairs for use in the invention include, but are not limited to, antigens/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, peptide/anti-peptide, ligand/receptor and rhodamine/anti-rhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin. Other suitable binding pairs include polypeptides such as the FLAG-peptide (DYKDDDDK; SEQ ID NO: 16) [Hopp et al., BioTechnology, 6:1204 1210 (1988)]; the KT3 epitope peptide (Martin et al., Science 255:192 194 (1992)); tubulin epitope peptide (Skinner et al., J. Biol. Chem 266:15163 15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393 6397 (1990)) and the antibodies each thereto. Generally, in a preferred embodiment, the smaller of the binding pair partners serves as the detectable label, as steric considerations may be important.
In one aspect, processes are provided herein for cleaving an oligonucleotide containing a dR-O—[C]n residue that forms a duplex with a nucleic acid, by contacting the duplex with a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases. In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot).
In some embodiments, the process further comprises the step of contacting the oligonucleotide with the nucleic acid to form the oligonucleotide/nucleic acid duplex. In some embodiments, this comprises hybridizing the oligonucleotide to the nucleic acid. In some embodiments, this comprises (i) contacting the oligonucleotide with a recombinase to form a recombinase/oligonucleotide complex; and (ii) contacting the recombinase/oligonucleotide complex to the nucleic acid to form the oligonucleotide/nucleic acid duplex. In some embodiments, the nucleic acid is the product of a nucleic acid amplification reaction (e.g., a recombinase polymerase amplification (RPA) process or a polymerase chain reaction (PCR)).
In some embodiments, the process further comprises the step of detecting cleavage of the oligonucleotide. In some embodiments, the detection is monitored in real time. In some embodiments, the detection is monitored at an endpoint for the reaction.
In some embodiments, the oligonucleotide contains a fluorophore and a quencher, where one of the fluorophore or the quencher is conjugated to the carbon linker. The nuclease activity excises the conjugated fluorophore or quencher from the oligonucleotide and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
In another aspect, processes are provided herein for detecting the presence or absence of a target nucleic acid. The processes comprise the following steps: (a) contacting an oligonucleotide probe containing a dR-O—[C]n residue or nucleotide with the target nucleic acid to form a probe/nucleic acid duplex; (b) contacting the duplex with a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases to excise the linker from the complex and/or specifically cleave the probe at the dR-O—[C]n nucleotide; and (c) detecting whether such excision or cleavage has occurred. In some embodiments, the nucleic acid is the product of a nucleic acid amplification reaction (e.g., a recombinase polymerase amplification (RPA) process or a polymerase chain reaction (PCR)). In some embodiments, the amplification reaction is monitored in real time. In some embodiments, the amplification reaction is monitored at an endpoint for the reaction.
In some embodiments, the duplex is formed by hybridizing the probe to the nucleic acid. In some embodiments, the duplex is formed by (i) contacting the probe with a recombinase to form a recombinase/probe complex; and (ii) contacting the recombinase/probe complex to the nucleic acid to form the probe/nucleic acid duplex.
In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot).
In some embodiments, the probe contains a fluorophore and a quencher, where one of the fluorophore or the quencher is conjugated to the carbon linker. For example, the fluorophore and the quencher are separated by 4-6 bases in the probe. In some embodiments, the fluorophore or the quencher that is not conjugated to the carbon linker is conjugated to the end (e.g., the 5′-end) of the probe. The nuclease activity excises and frees the conjugated fluorophore or quencher associated with the dR-O—[C]n residue from the probe and the detection step comprises measuring a difference, if any, in fluorescence between the conjugated and free state.
In another aspect, provided herein are oligonucleotide probes containing a dR-O—[C]n residue. In some embodiments, the probes are 30 to 60 nucleotides in length and contain a fluorophore quencher pair separated by 10 nucleotides or less (e.g., 4-6 nucleotides), where either the flurophore or the quencher is conjugated to the dR-O—[C]n residue. In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the fluorophore or the quencher that is not conjugated to the dR-O—[C]n residue is conjugated to the end (e.g., the 5′-end) of the probe. In some embodiments, the probes are 30 to 40 nucleotides (e.g., 35 nucleotides) in length.
In yet another aspect, provided herein are kits comprising (i) an oligonucleotide containing a dR-O—[C]n residue, and (ii) a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases. In some embodiments, the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg).
In yet a further aspect, provided herein are reaction mixtures comprising an oligonucleotide containing a dR-O—[C]n residue and a nuclease selected from an AP endonucleases, or DNA glycosylases, or an DNA glycosylase/lyases (e.g., endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg)). In some embodiments, the linker is a 3-6 carbon atom linker (e.g., a 6 carbon atom linker). In some embodiments, the oligonucleotide is blocked at its 3′-end to prevent polymerase extension. In some embodiments, the linker is conjugated to a detectable label (e.g., biotin, digoxygenin, peptide, fluorophore, quencher, antibody or a quantum dot). In some embodiments, the reaction mixture is freeze dried or lyophilized.
In some embodiments, the reaction mixture further comprises a container. For example, the reaction mixture can be contained in a tube or in a well of a multi-well container. The reaction mixtures may be dried or attached onto a mobile solid support such as a bead or a strip, or a well.
In some embodiments, the reaction mixture further comprises a target or template nucleic acid that contains a sequence that is complementary to the oligonucleotide.
FIG. 2 indicates both primary sequence and schematically the nature of a dR-biotin probe generated for use in an RPA DNA amplification reaction using as a target a DNA molecule containing the sequence specified in the probe. The probe is blocked (to prevent polymerase extension during the amplification phase) and contains an internal dR-biotin as the test substrate for the enzymes. The probe also contains a 5′-FAM. Thus, in principle, if DNA is amplified in a reaction containing this probe there is the possibility that the probe will bind to and interact specifically with the amplified DNA either by ‘classical’ hybridization to complementary single strands formed during amplification, or by recombinase-mediated processes. The outcome of such an experiment is shown in FIG. 3 and described in Example 1 below.
A second set of experiments was performed to investigate the generality of this cleavage activity, and in this case using fluorescent reagents in which the dR-O—[C]n nucleotide is coupled to a fluorophore as depicted in FIG. 4. In this case the dR-fluorophore is positioned close to the 5′ end of the oligonucleotide probe and in close proximity to a quencher which is attached to the very 5′ end. As before the 3′ end of the probe is suitably blocked to prevent aberrant elongation or primer artefacts. As indicated in FIG. 4, should the probe form hybrids with complementary amplifying material then it might become a substrate for fpg (or Nfo) and if so could cleave the backbone at this position (and potentially release the fluorophore directly into the aqueous medium detached from either oliogonucleotide fragment if the glycosylase activity is present in fpg or other non-AP endonuclease enzymes). If cleavage occurs there will be physical separation of the fluorophore and quencher and hence an increase in detectable fluorescence in a manner akin to that described earlier for THF-based fluorescent probes utilising E. coli Nfo or exoIII proteins. FIGS. 5 and 6 show the outcome of such experiments and describe in Example 2 in which RPA reactions were performed on human genomic targets utilizing primers and probes specifically directed toward known single nucleotide polymorphism (SNP) regions.
These experiments collectively clearly demonstrate that dR-O—[C]n groups are substrates for the Nfo and the fpg nucleases. Furthermore, it is possible to construct probes containing such groups in a way that the activity of the nucleases on the probe occurs only in the circumstance that complementary nucleic acid strands accumulate permitting duplex formation, thereby allowing determination of whether the amplification has occurred by fluorescence or other mechanisms. Therefore, these dR-O—[C]n nucleotide reagents could be broadly applied in combination with fpg, Nfo or glycosylase/lyase and equivalent enzymes for a variety of uses.
All sequence citations, references, patents, patent applications or other documents cited are hereby incorporated by reference.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
Example 1
Oligonucleotides Probes Containing an Internal dR-Biotin are Cut by Nfo and Fpg
In this example it is shown that oligonucleotides probes, depicted schematically in FIG. 2, containing an internal dR-Biotin can be cut by Nfo and fpg. The reactions (total volume of 150 μL) were mixed from fresh reagents and incubated for 75 minutes at 37° C. Conditions used were 50 mM Tris/Acetate (pH 7.9), 14 mM Mg-Acetate, 100 mM Potassium-Acetate, 2 mM DTT, 200 nM each dNTP, 6% PEG 35,000, 3 mM ATP, 50 mM Phospho-Creatine, 900 ng/μL T4gp32, 120 ng/μL, T4uvsX, 30 ng/μL T4uvsY, 360 ng/μL Bac. subtilis DNA polI. Either 3000 copies of DNA template or water (as a negative control) was included as indicated. Nuclease, 200 ng/μL Nfo or 50 ng/μL fpg, was included as indicated. Primers, K2 and J1, were included at 480 nM each and the probe, FpgProb1, was included at 120 nM, with their sequences provided below. Samples were quenched in one volume of 2% SDS/one volume phenol, mixed and incubated for 20 minutes at 65° C. Subsequently samples were phenol/chloroform extracted and twice ethanol precipitated according to standard molecular biology techniques. Half of each sample was then resuspended in formamide loading buffer resolved on a 16.5% denaturing polyacrylamide gel (Urea) and visualised (using the FAM fluorescence) following standard protocols. Markers were 2 pmol of the probe and 2 pmol of a 32 nt marker oligonucleotide.







J1


(SEQ ID NO: 1)


5′-acggcattaacaaacgaactgattcatctgcttgg-3′


 


K2


(SEQ ID NO: 2)


5′-ccttaatttctccgagaacttcatattcaagcgtc-3′


 


FpgProbe1


(SEQ ID NO: 3)


5′-6FAM-cagaagtatgaccgtgtctttgaaatg[dR-biotin]


ttgaagaaatggtt[ddC]-3′






The probe and any derivatives were visualised here by virtue of the FAM moiety which emits visible light when excited by UV radiation. Amplification reactions (RPA) containing a target DNA, two (2) appropriate amplification primers, the dR-biotin probe and either no nuclease, Nfo protein, or fpg protein were cleaned following incubation and separated by size on a denaturing acrylamide gel and then exposed to UV. The probe or any derivatives retaining the 5′-FAM are then visible (FIG. 3). In the absence of an added nuclease, the probe, 42 nucleotides long, mostly migrates at its expected location slightly more slowly than a control labelled primer of 32 nucleotides (indicated). A slightly slower-migrating (longer) fragment is also seen as compared to the neat probe not incubated in RPA (#1). This likely arose because the probe can be unblocked slowly by nucleases that are believed to be present in some of the enzyme preparations (nibbling at the 3′ end), and once unblocked it can be extended following hybridization to amplifying target and hence forming a nested amplicon of sorts. In the presence of Nfo however, this phenomenon is much more prominent as expected and a large proportion of the probe is now elongated. Furthermore, the Nfo protein was indeed attacking the dR-O—[C]n residue rather than just ‘polishing’ the 3′ end because some small amount of faster-migrating probe DNA (#2) is also visible indicating cleavage at the dR-O—[C]n location with no subsequent elongation. Finally, when fpg protein was included in the reaction environment a large proportion of faster-migrating cleaved probe is visible and no elongated material is detected, as fpg leaves a blocked 3′-end after cleavage and hence it is not extended by polymerase enzyme present in the mix.
Example 2
Measurement of DNA Amplification with Oligonucleotides Probes Containing an Internal dR-Fluorophore
In this Example, RPA experiments using fluorescent reagents in which the dR-O—[C]n nucleotide is coupled to a fluorophore as depicted in FIG. 4. In this case, the dR-fluorophore is positioned close to the 5′ end of the oligonucleotide probe and in close proximity to a quencher which is attached to the very 5′ end. As in the previous example, the 3′ end of the probe is suitably blocked to prevent aberrant elongation or primer artefacts.
The reactions (total volume of 50 μL) were performed according to standard RPA protocol for freeze-dried reactions. Briefly, lyophilised reagents were mixed with PEG, Magnesium-Acetate and template, and incubated for 20 minutes at 38° C. in a fluorometer (Twista prototype; ESE GmbH, Germany). Conditions used were 50 mM Tris/Acetate (pH 8.3), 14 mM Mg-Acetate, 100 mM Potassium-Acetate, 5 mM DTT, 240 nM each dNTP, 5% PEG 35,000, 4% Trehalose 2.5 mM ATP, 50 mM Phospho-Creatine, 300 ng/μL rb69gp32, 273 ng/μL uvsX, 120 ng/μL uvsY, 50 ng/μL Staph. aureus DNA polI. For the experiments of FIG. 5, 1000, 100, 10 or 0 copies of the DNA template was included as indicated in the figure, while 1000 copies of the DNA template was included for the experiments of FIG. 6. Fpg nuclease, 25 ng/μL, was included. Primers were included at 360 nM each and probe was included at 120 nM, with their sequences provided below. Fluorescence was measured every 20 seconds (excitation 470 nM, emission 520 nM). Samples were removed from the incubator for a brief mix/spin at 4 minutes of incubation time and returned to the incubator/fluorometer. Arbitrary fluorescence units were plotted against time in minutes.
For human genomic locus rs4824871 the sequences of the primers, F2 and R1, and the probe used were:







F2


(SEQ ID NO: 4)


5′-ccatcctcaatactaagctaagtaaaaagattt-3′


 


R1


(SEQ ID NO: 5)


5′-ccctgtggctaagagctcttgatagtcaaagta-3′


 


Probe


(SEQ ID NO: 6)


BHQ1-5′-cctt[dR-FAM]tctaaggaaatggacag


aaataggcaagat[ddC]-3′






For human genomic locus rs1207445 the sequences of the primers, F2 and R2, and the probe used were:







F2


(SEQ ID NO: 7)


5′-cccttctgatattctaccaaatgccccctaaat-3′


 


R2


(SEQ ID NO: 8)


5′-catgtgtataagaaaactacccaagcctaggga-3′


 


Probe


(SEQ ID NO: 9)


BHQ1-5′-cagtg[dR-FAM]ccaatacacacacac


aagactgggcatgg[ddC]-3′






For human genomic locus rs1105561 the sequences of the primers, F1 and R1, and the probe used were:









F1



(SEQ ID NO: 10)



5′-tatagtggaaaggtgttcatttgtataaacccc-3′



 



R1



(SEQ ID NO: 11)



5′-cacataaatcagagaatgtgtggggtcatgtat-3′



 



Probe



(SEQ ID NO: 12)



BHQ1-5′-aactt[dR-FAM]gcaactaacgctaaa



ttataatcacttct[ddC]-3′






For human genomic locus rs5923931 the sequences of the primers, F1 and R1, and the probe used were:









F1



(SEQ ID NO: 13)



5′-catttctcaaaagaagatatgcaaataaaaaca-3′



 



R1



(SEQ ID NO: 14)



5′-ccattataactggggtgagatgatatctcattg-3′



 



Probe



(SEQ ID NO: 15)



BHQ1-5′-tctca[dR-FAM]cataactgatcatcag



agaaatgtaaatc[ddC]-3′






FIGS. 5 and 6 show the outcome of the above experiments in which RPA reactions were performed on human genomic targets utilizing primers and probes specifically directed toward known SNP regions. In FIG. 5 two such genomic regions were amplified using RPA and probes with the general structure depicted in FIG. 4, along with the inclusion of the fpg protein. Target genomic DNA has been added to give a total target copy number of 1000, 100, 10 or zero target molecules, and in this way the requirement for the specific accumulation of amplicons matching the target is ensured. Note that after between 6 and 12 minutes (depending on the target and copy number) there is a clear rise in fluorescence in those samples containing target, whilst those lacking targets remain with more-or-less stable fluorescence. In FIG. 6 there is similar data shown for four human genomic DNA target/probe sets (two of which were also used in FIG. 5) and in each case fluorescence rises at about the expected time of DNA amplification. In addition to these and other successful probes, we have encountered occasional probes that did not seem to work well in RPA amplification/detection systems (maybe 10-20% of those analyzed) however the source of these failures is as yet unclear, potentially reflecting failures in RPA amplification in some cases rather than probe failure, potentially as a result of probe failure in other cases. We speculate that in some cases the position and nature of adjacent bases, or the nature of the base opposing the dR-O—[C]n group could play a part in the effectiveness of the probe.
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. Other aspects, advantages, and modifications considered to be within the scope of the following claims. The claims presented are representative of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.

## Claims

1. An oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising:
a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms, wherein said oligonucleotide contains a fluorophore-quencher pair separated by 10 nucleotides or less,
wherein the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8.

2. The probe of claim 1, wherein n is 3-6.

3. The probe of claim 2, wherein n is 6.

4. The probe of claim 1, wherein the fluorophore and quencher are separated by 4-6 bases.

5. The probe of claim 1, wherein the fluorophore or quencher not conjugated to the dR-O—[C]n nucleotide is conjugated at one end of the probe.

6. The probe of claim 5, wherein the conjugated end of the probe is the 5′-end.

7. A kit, comprising
(i) an oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms,
wherein the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8; and
(ii) 8 oxoguanine DNA glycosylase (fpg).

8. The kit of claim 7, wherein n is 3-6.

9. The kit of claim 7, wherein n is 6.

10. A reaction mixture comprising
an oligonucleotide having a first 5′-end and a first extension blocked 3′-end, the oligonucleotide comprising a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms,
wherein the dR-O—[C]n nucleotide is conjugated with either the fluorophore or the quencher,
wherein the oligonucleotide is between 30 and 60 nucleotides in length,
wherein cleavage of the oligonucleotide by 8-oxoguanine DNA glycosylase yields (i) a first fragment comprising the first 5′-end and a second extension blocked 3′-end and (ii) a second fragment comprising a second 5′-end and the first extension blocked 3′-end, and
wherein n is 1-8; and
8-oxoguanine DNA glycosylase (fpg).

11. The reaction mixture of claim 10 wherein n is 3-6.

12. The reaction mixture of claim 11, wherein n is 6.

13. The reaction mixture of claim 10, wherein the reaction mixture is lyophilized.

14. The reaction mixture of claim 10, further comprising a container.

15. The reaction mixture according to claim 14, wherein the container is a tube.

16. The reaction mixture of claim 10, further comprising a target nucleic acid that contains a sequence complementary to the oligonucleotide.