EP4314820A1

EP4314820A1 - Analyte capture

Info

Publication number: EP4314820A1
Application number: EP22712620.8A
Authority: EP
Inventors: Tim Albrecht; Oliver IRVING
Original assignee: University of Birmingham
Current assignee: University of Birmingham
Priority date: 2021-03-25
Filing date: 2022-03-24
Publication date: 2024-02-07
Also published as: WO2022200793A1

Abstract

The invention relates to a method of synthesising a modular polynucleotide, in one reaction, from two or more subunits that are each less than 100 base pairs in length.

Description

ANALYTE CAPTURE

Field of the Invention

The invention relates to a modular polynucleotide and to a method of making a modular polynucleotide. The invention also relates to a method and a kit for determining if one or more analyte(s) is/are present in a sample, and a method and kit for diagnosis of a medical condition, using a modular polynucleotide. The invention also extends to a method of treatment, and a method of determining the efficacy of a therapeutic agent.

Background

Assays are an important aspect of investigative research as they enable one to measure the presence, amount and/or functional activity of an analyte. Each analyte of interest may require a different specialised technique or equipment to detect its presence. For example, a sample suspected of containing several different analytes may require the use of gas chromatography-mass spectrometry (GC-MS) or high-pressure liquid chromatography-mass spectrometry (HPLC-MS) to detect analytes present at low concentrations; enzyme linked immunosorbent assays (ELISA) or immunofluorescence (IF) to detect analytes in the form of polypeptides or proteins; and polymerase chain reaction (PCR) or a microarray to detect nucleic acids. While each technique enables detection and/or quantification of specific types of analytes, each technique is often also expensive and/or time-consuming. There is therefore a need for a rapid and cheap technology for detecting several different analytes. Statements of the Invention

The inventors have surprisingly developed a novel method of creating a modular double-stranded polynucleotide having subunits (i.e., (im)mature conjugate subunits) each comprising a probe for binding an analyte. The modular polynucleotide can be used with a carrier-enhanced resistive pulse sensing technology (e.g., nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing) to simultaneously detect several different analytes, including different types of analyte, in a high- throughput manner.

Thus, according to a first aspect of the invention, there is provided a method of making an immature conjugate subunit, the method comprising: a) conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit.

In one embodiment, there is provided a method of making a mature conjugate subunit, the method comprising: a) conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit; and b) forming a (first) mature conjugate subunit by cleaving the double-stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end, or a first sticky end and/or a second sticky end.

The first sticky end and/or the second sticky end of the mature conjugate subunit may be complementary to a (first or second) sticky end of a further, separate mature conjugate subunit, or complementary to a (first or second) sticky end of a further, separate mature conjugate subunit to be formed from an immature conjugate subunit.

Each mature conjugate subunit may further comprise a double-stranded polynucleotide spacer. The spacer may be annealed to the double-stranded polynucleotide of the (first) mature conjugate subunit so as to create a polynucleotide backbone. Thus, the method according to the first aspect may further comprise the step of: c) annealing the first sticky end or the second sticky end of the (first) mature conjugate subunit to a complementary sticky end of a (first) double-stranded polynucleotide spacer.

The invention may further comprise creating a modular polynucleotide comprising a double-stranded polynucleotide backbone having two or more mature conjugate subunits. Thus, the method according to the first aspect may further comprise:

• repeating steps (a) to (b) or steps (a) to (c) to create a total of two or more mature conjugate subunits, wherein each of the two or more mature conjugate subunits has a (first or second) sticky end that is complementary with a (first or second) sticky end of a separate mature conjugate subunit; and • annealing the sticky ends of the two or more mature conjugate subunits to create a modular polynucleotide comprising a double-stranded polynucleotide backbone having two or more mature conjugate subunits.

The method of creating a modular polynucleotide may comprise performing a one-pot synthesis reaction. In other words, the cleaving and annealing steps may be performed simultaneously in a single reaction vessel. Thus, the modular polynucleotide may be created by placing multiple immature conjugate subunits in a single reaction vessel together with an exonuclease, a DNA ligase and a DNA polymerase, and optionally multiple double-stranded polynucleotide spacers.

The modular polynucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more, 3000 or more, 5000 or more, 10000 or more mature conjugate subunits. The modular polynucleotide may comprise between two and 10000 mature conjugate subunits, two and 5000 mature conjugate subunits, two and 1000 mature conjugate subunits, two and 500 mature conjugate subunits, two and 100 mature conjugate subunits, two and 50 mature conjugate subunits, or two and 10 mature conjugate subunits.

The method of the invention may ultimately comprise attaching a plurality of mature conjugate subunits (each comprising a probe) together in a modular fashion. The initial polynucleotide used to create the mature conjugate subunits may be a blunt - ended polynucleotide. Prior art methods of attaching blunt-ended polynucleotides together can only be achieved with polynucleotides that are at least 100 base pairs in length (and by using an exonuclease, a DNA polymerase and a DNA ligase). However, unlike the prior art, the method according to the invention can be used to attach several different blunt-ended polynucleotides together that are significantly shorter in length (e.g., blunt-ended polynucleotides that are between about 20 and about 100 base pairs in length or between about 34 and about 100 base pairs in length).

The inventors believe (but do not wish to be bound by the theory) that the attachment of a probe to the blunt-ended polynucleotide sterically impedes the activity of nucleases (e.g., exonucleases) that may be used to cleave short (i.e., less than 100 base pairs) blunt-ended double-stranded polynucleotides (e.g., immature conjugate subunits). They believe that nucleases (such as exonucleases, e.g., T5 exonuclease) are unable to navigate across the site at which the steric hindering agent is located (e.g., the probe), and thus get knocked off of the blunt-ended polynucleotide. Consequently, the method according to the invention enables the blunt ends of a short double-stranded polynucleotide to be converted into “sticky ends” without converting the entire double-stranded polynucleotide into a single-stranded polynucleotide.

The method according to the invention is also advantageous because it enables probes for different analytes, different classes of analyte and the same analyte to be conjugated to a single polynucleotide backbone of a modular polynucleotide. Consequently, a modular polynucleotide according to the invention can be used to perform multiplex, high-throughput analysis using a single technique (i.e., carrier- enhanced resistive pulse sensing, such as nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing).

Furthermore, the method according to the invention enables each probe of a modular polynucleotide to be conjugated at a specific location within the polynucleotide backbone. Consequently, each disruption of the electrical current generated during use with a carrier-enhanced resistive pulse sensing technology can be attributed to a particular probe, and the specific characteristics of each disruption can be used to determine if a probe has bound an analyte or not.

Furthermore, the method can be used to easily modify a modular polynucleotide so as to alter the analyte(s) being detected due to the modular nature of the immature conjugate subunits, which may each comprise an optional double-stranded polynucleotide spacer.

The probe conjugation site referred to herein may comprise a nucleotide sequence specific for a transferase enzyme. Thus, the probe conjugation site may comprise or consist of a CpG island (or a CpG thereof) or the nucleotide sequence TCGA. In one embodiment, the probe conjugation site comprises a nucleotide sequence specific for a methyl transferase enzyme (e.g., m.Taql). Thus, the probe conjugation site may comprise the nucleotide sequence TCGA, or a CpG island (or a CpG thereof). A double-stranded (im)mature conjugate subunit may be created by a method according to the invention.

Thus, according to a second aspect, there is provided an immature conjugate subunit comprising: a probe, for binding an analyte, conjugated to a probe conjugation site of a double-stranded polynucleotide, wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme.

The polynucleotide of the double-stranded mature conjugate subunit referred to herein may be between about 20 and about 100 base pairs/nucleotides in length, or between about 34 and about 100 base pairs/nucleotides in length.

The polynucleotide of the double-stranded mature conjugate subunit may comprise blunt ends (e.g., a first blunt end and/or a second blunt end).

The polynucleotide of the double-stranded mature conjugate subunit may comprise sticky ends (e.g., 5’-sticky ends or 3’-sticky ends). Thus, the conjugate subunit may be referred to as a mature conjugate subunit.

A double-stranded modular polynucleotide may be created by a method according to the invention. For example, a modular polynucleotide may be created by annealing a plurality of mature or immature conjugate subunits together. Each of the immature and mature conjugate subunits may be created by a method according to the invention.

Thus, according to a third aspect, there is provided a double-stranded modular polynucleotide comprising: a double-stranded polynucleotide backbone comprising a plurality of probe conjugation sites each site having a nucleotide sequence specific for a transferase enzyme, wherein each probe conjugation site is separated by at least 16 base pairs, and wherein at least one probe conjugation site is conjugated to a single probe for binding an analyte. Of the plurality of probe conjugation sites, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the sites may be conjugated to a probe. 100% of the probe conjugation sites may be conjugated to a probe.

The modular polynucleotide may comprise different classes of probe, which are capable of binding to the same analyte or different analytes. The modular polynucleotide may comprise two or more different classes of probe, which are capable of binding to the same analyte or a different analyte.

The (im)mature conjugate subunit according to the second aspect may be used to form a modular polynucleotide according to the invention (e.g., the third aspect of invention) or a modular polynucleotide made by a method according to the invention. A single modular polynucleotide according to the invention may be used to perform multiplex, high-throughput analysis using a carrier-enhanced resistive pulse sensing technology to simultaneously detect different analytes using different probes.

The method according to the invention is also advantageous because it can be used to create a modular polynucleotide comprising a plurality of identical probes. None, some or all of the probes of such a modular polynucleotide may be capable of binding to relevant analyte molecules in a sample. Thus, such a modular polynucleotide can be used to improve the signal -to-noise ratio and/or reduce the total analysis time. The modular polynucleotide can also be used to provide quantitative information about analyte binding (analyte concentration in a sample).

The probe conjugation site referred to herein may comprise a nucleotide sequence specific for a transferase enzyme (e.g., a methyl transferase enzyme). Thus, the probe conjugation site may comprise or consist of a CpG island (or a CpG thereof) or a “TCGA” nucleotide sequence as they are specific for methyl transferases. In one embodiment, the probe conjugation site comprises a nucleotide sequence specific for a methyl transferase enzyme (e.g., m.Taql). Thus, the probe conjugation site may comprise the nucleotide sequence TCGA. The methyl transferase enzyme may be an enzyme that uses S-adenosyl methionine (SAM) as a starting reagent. In other words, the methyl transferase enzyme may be an enzyme that binds SAM.

Each of the probe conjugation sites referred to herein may be separated by at least about 15 base pairs, at least about 30 base pairs, at least about 100 base pairs, or at least about 300 base pairs. The greater the distance between each probe conjugation site, the easier it is to detect a conjugated probe or an analyte bound to a conjugate probe. Most preferably each of the probe conjugation sites is separated by between about 300 base pairs and about 1000 base pairs.

The inventors have surprisingly demonstrated that a modular polynucleotide according to the invention can be created (see Examples 1 and 2). It is a simple and robust solution to investigate a diverse range of analytes (e.g., biomarkers) quickly, simultaneously and at a low cost. Modular polynucleotides can be flexibly designed to carry a range of different probes, or multiple identical probes, depending on the application. Furthermore, the invention provides a novel way of creating modular polynucleotides with improved efficiency, in terms of time, cost and design flexibility.

The skilled person would appreciate that the conjugation of one or more probes to a polynucleotide may be determined by a carrier-enhanced resistive pulse sensing technology (see Loh et al., Anal. Chem. 2018, 90 (23): 14063-14071). Thus, the skilled person would also appreciate that modular polynucleotides and (im)mature conjugate subunits according to the invention (or made by methods according to the invention) may be used with a carrier-enhanced resistive pulse sensing technology to detect the binding of analytes in a sample.

According to another aspect of the invention, there is provided a method of creating a recombinant polynucleotide, the method comprising:

(a) (i) using a method according to the invention to create a modular polynucleotide comprising one or more probes each conjugated to a probe conjugation site of a polynucleotide backbone via a UV-sensitive bond, or

(ii) providing a modular polynucleotide comprising one or more probes each conjugated to a probe conjugation site of a polynucleotide backbone via a UV- sensitive bond; and (b) creating a recombinant polynucleotide by exposing the modular polynucleotide by to UV light in order to cleave the UV-sensitive bond(s) of the modular polynucleotide.

The method according to the invention is advantageous because it would enable the creation of specific sequences that can bridge two or more double-stranded polynucleotides together without the use of a specific restriction site or a large insert. Consequently, less “junk” DNA and more coding DNA can be inserted into viral vectors. Viral vectors have a limited capacity for inserts DNA.

In another aspect, there is provided a method of creating sticky ends on a blunt-ended polynucleotide, the method comprising: a) (i) conjugating a steric hindering agent to a conjugation site of a polynucleotide comprising a first blunt end and/or a second blunt end, or

(ii) providing a polynucleotide comprising a first blunt end and/or a second blunt end and a steric hindering agent conjugated to a conjugation site of the double-stranded polynucleotide.

The method may optionally further comprise: b) cleaving the first blunt end and/or the second blunt end of the polynucleotide using a nuclease to create a first sticky end and/or a second sticky end.

The steric hindering agent may be a probe as defined herein. The steric hindering agent impedes the activity of nucleases (e.g., exonucleases) that may be used to cleave short (i.e., less than 100 base pairs) blunt-ended double-stranded polynucleotides (e.g., an immature conjugate subunit). The inventors believe that the conjugation of the steric hindering agent to the conjugation site prevents that exonuclease from continuing its cleavage activity along the entire length of the polynucleotide comprising the sticky end(s). Consequently, the method according to the invention enables the blunt ends of a short double-stranded polynucleotide to be converted into “sticky ends” without converting the entire double-stranded polynucleotide into a single-stranded polynucleotide.

Each probe may be conjugated to a separate polynucleotide using any method known in the art. The conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an adaptor to facilitate conjugation of the probe to the polynucleotide. Thus, the conjugating step may comprise chemically- modifying or functionalising the probe conjugation site (e.g., TCGA or a CpG of a CpG island) of the double-stranded polynucleotide with an adaptor. One or more, two or more, three or more, or four more nucleotides of the probe conjugation site may be chemically modified. Specifically, the conjugating step may comprise chemically- modifying or functionalising the probe conjugation site of the polynucleotide with an azide (N3) group or a peptide adaptor. Preferably the conjugating step comprises chemically attaching an azide group to the probe conjugation site. Preferably the nucleotides of the probe conjugation site are not terminal base-paired nucleotides (i.e., nucleotides located within four nucleotides of the 5’-basepaired terminus or the 3’- basepaired terminus of the polynucleotide). Preferably the probe conjugation site is at least 15 nucleotides from the 5 ’-terminus and the 3 ’-terminus of the polynucleotide.

The conjugating step may comprise attaching an azide group to the polynucleotide by contacting the polynucleotide with an azide donor. The azide donor may be a substance comprising an azide group and that has the ability to donate the azide group without reacting to other groups on the recipient molecule. The azide donor may be RAdoHcy-8-Hy-PEG-N3, a modified SAM molecule containing an azide group (rather than a methyl) or an azide modified nucleotide. Preferably the azide donor is RAdoHcy-8-Hy-PEG-N3 or a modified SAM molecule containing an azide group. The conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an azide (-N3) group by contacting the polynucleotide with an azide donor (e.g., RAdoHcy-8-Hy-PEG-N3 or a SAM molecule containing an azide group) in the presence of a catalyst, such as an enzyme. The enzyme may be a methyl transferase. Preferably the methyl transferase is Taql methyl transferase (EC

2.1.1.72). The conjugating step may comprise chemically-modifying or functionalising the polynucleotide with an azide (N3) group by contacting the polynucleotide with an azide donor (e.g., RAdoHcy-8-Hy-PEG-N3 or a SAM molecule containing an azide group) in the presence of a methyl transferase, such as Taql methyl transferase (EC

2.1.1.72) for about 30 minutes to about 90 minutes at about 50°C.

The conjugating step may comprise chemically-modifying or functionalising the probe to facilitate conjugation to the polynucleotide without interfering with the ability of the probe to bind an analyte. Thus, the skilled person would appreciate that the probe should be functionalised in a region that is not responsible for binding to an analyte. Thus, in embodiments in which the probe is an antibody, the antibody should not be functionalised in or near the antibody binding region (e.g., Fab). The antibody should be functionalised in a region such as the Fc region. For example, in embodiments where the probe is an antibody, the terminus of Fc region (e.g., C_H3 or C_H2) of the antibody is functionalised. In embodiments where the probe is a scFv antibody, the linker is functionalised. In embodiments where the probe is a single stranded DNA/ RNA, the 5 ’end of the single stranded DNA/RNA is functionalised with an amine group for conjugation. In embodiments where the probe is a single-stranded polynucleotide, the 3 ’end is functionalised.

The conjugating step may comprise conjugating the probe directly or indirectly to the polynucleotide. The conjugating step may comprise covalently conjugating the probe directly to the polynucleotide. The conjugating step may comprise conjugating the probe to the polynucleotide via a photocleavable bond (e.g., a UV-sensitive bond). The conjugating step may comprise conjugating (e.g., covalently conjugating) the probe directly or indirectly to the polynucleotide conjugation site. Preferably the probe is not conjugated to a terminal base-paired nucleotide. Preferably the probe conjugation site is not located within 15 base pairs from the 5 ’-terminus or the 3’- terminus of the polynucleotide. The probe may be conjugated to a polynucleotide that has been chemically-modified or functionalised. The probe may be conjugated to a functional group or chemical group of a nucleotide that has been chemically-modified or functionalised. The conjugating step may comprise conjugating the probe to the double-stranded polynucleotide via a linker (e.g., DBCO-NHS ester, a peptide nucleic acid, a histidine tag). The conjugating step may comprise covalently conjugating the probe to the double-stranded polynucleotide via a linker (e.g., DBCO-NHS ester, a peptide nucleic acid or a histidine tag). Thus, the conjugating step may comprise chemically-modifying or functionalising the probe with a DBCO-NHS ester, a peptide nucleic acid or a histidine tag. Preferably the conjugating step comprises chemically- modifying or functionalising the probe with a DBCO-NHS ester. Thus, the conjugating step may comprise covalently conjugating the probe to the double- stranded polynucleotide via a DBCO-NHS ester in the presence of a salt solution in order to increase conjugation efficiency. The salt solution may have a concentration of about 0.1M or greater. The salt solution may have a concentration of about 0.1M to about 0.2M. The conjugating step may comprise chemically-modifying or functionalising a terminal monomer of the probe with a DBCO-NHS ester. The conjugating step may comprise chemically-modifying or functionalising a terminal monomer with an amine group. The conjugating step may comprise chemically- modifying or functionalising a terminal amine group of the probe with a DBCO-NHS ester. The conjugating step may comprise chemically-modifying or functionalising the probe with an amine group, followed by functionalising the amine group with a DBCO-NHS ester. The skilled person would appreciate how to functionalise a variety of different types of probes with an amine group, particularly the terminal monomer of the probe. The conjugating step may comprise chemically-modifying or functionalising the probe by contacting it with a DBCO-NHS ester. The conjugating step may comprise chemically-modifying or functionalising a terminal amine group of the probe by contacting it with a DBCO-NHS ester. The probe or amine group of the probe may be contact with the DBCO-NHS ester at group at about 18°C to 25°C for about 2 hours to about 16 hours.

Thus, the conjugating step may comprise contacting a functionalised polynucleotide with a functionalised probe. In one embodiment, the conjugating step comprises contacting a polynucleotide functionalised with a peptide adaptor with a probe functionalised with a PNA or a histidine tag (e.g., a histidine-tagged antibody), in order to conjugate the probe to the polynucleotide. In another embodiment, the conjugating step comprises contacting a polynucleotide functionalised or chemically modified with an azide group with a probe functionalised with a DBCO-NHS ester, in order to conjugate the probe to the polynucleotide. The probe conjugation site of the polynucleotide may be functionalised with an azide group. The conjugating step may comprise contacting a polynucleotide functionalised with an azide group with a functionalised probe in an azide-alkyne reaction, preferably a copper-free azide- alkyne reaction. The polynucleotide functionalised with an azide group may be contacted with a probe functionalised with a DBCO-NHS ester at about 18°C to 25°C for about 1 hour to about 2 hours, or at about 2°C to about 6°C for about 16 hours to 24 hours. Preferably the polynucleotide functionalised with an azide group is contacted with a probe functionalised with a DBCO-NHS ester at about 18°C to 25°C for about 1.5 hours, or at about 4°C for about 16 hour to 24 hours. The conjugating step may comprise conjugating the probe to a probe conjugation site of the double-stranded polynucleotide via a photocleavable bond (e.g., a UV-sensitive bond).

The double-stranded polynucleotide of the (im)mature conjugate subunit may be a polynucleotide comprising artificial nucleotides or natural polynucleotides. One or more of the nucleotides may comprise an epigenetic modification, such as a methylation. The double-stranded polynucleotide may be DNA. Thus, the polynucleotide may be DNA comprising a probe conjugation site. The probe conjugation site may comprise or consist of a nucleotide sequence specific for a transferase enzyme (e.g., the nucleotides TCGA, or a CpG of a CpG island).

The probe conjugation site may be positioned at least 8 base pairs/nucleotides from the 5 ’-terminal nucleotide/base pair and at least 8 base pairs/nucleotides from the 3’- terminal nucleotide/base pair. The probe conjugation site may be positioned at least about 15 nucleotides from the 5’-terminus and/or the 3’-terminus; at least about 20 nucleotides from the 5 ’-terminus and/or the 3 ’-terminus; at least about 25 nucleotides from the 5 ’-terminus and/or the 3 ’-terminus; at least about 30 nucleotides from the 5’- terminus and/or the 3 ’-terminus; at least about 35 nucleotides from the 5 ’-terminus and/or the 3 ’-terminus; at least about 40 nucleotides from the 5 ’-terminus and/or the 3 ’-terminus; or at least about 45 nucleotides from the 5 ’-terminus and/or the 3’- terminus. The probe conjugation site sequence may be positioned between about 15 nucleotides and about 48 nucleotides from the 5 ’-terminus and the 3 ’-terminus. Preferably the probe conjugation site sequence is positioned about 30 nucleotides from the 5 ’-terminus and at least 30 nucleotides from the 3 ’-terminus.

The double-stranded polynucleotide may be between about 20 and about 100 base pairs/nucleotides in length or between about 34 and about 100 base pairs/nucleotides in length. Preferably the double-stranded polynucleotide is about 60 base pairs/nucleotides in length.

The double-stranded polynucleotide of the immature conjugate subunit may comprise blunt ends (e.g., a first blunt end and/or a second blunt end). The double-stranded polynucleotide of the mature conjugate subunit may comprise sticky ends (e.g., 5’- sticky ends or 3 ’-sticky ends). The probe may be any agent that selectively or specifically binds to an analyte. The probe may bind selectively or specifically to an analyte. Preferably the probe binds specifically to an analyte. Each or two or more of the probes of the modular polynucleotide may be capable of binding to the same analyte or may be identical. Each or two or more of the probes of the modular polynucleotide may be capable of binding to a different analyte.

The probe may be any probe known in the art. The probe may be a polymer (e.g., a ssRNA, a morpholino or a peptide nucleic acid). There are different classes of probe known in the art, such as polypeptides, proteins and polynucleotides. Thus, the probe may be one or more members selected from the group comprising a polypeptide, a protein (e.g., an antibody or an affimer), a polynucleotide (e.g., DNA or single- stranded DNA), a nanoparticle and an aptamer. Preferably, the probe(s) is/are one or more selected from the group consisting of a single-stranded nucleotide, an antibody, a (functional) fragment of an antibody and an aptamer.

The probe may be DNA, preferably single-stranded DNA, or RNA, preferably single- stranded DNA.

The probe may be an antibody or a (functional) fragment thereof (e.g., a scFv, a VL, a VH, a Fd; an Fv, an Fab, a Fab', a F(ab')2, an Fc fragment, or a bispecific antibody) that binds to an analyte. For example, when the invention is being used to detect sepsis biomarkers in a sample, the probe may be an antibody that binds to the sepsis biomarkers Interleukin 6 (IF-6) or Procalcitonin.

The term "antigen-binding region" can mean a region of the antibody having specific binding affinity for its target antigen/analyte. The binding region may be a hypervariable CDR or a functional portion thereof. The term “functional portion ” of a CDR can mean a sequence within the CDR which shows specific affinity for the target analyte .

The term "(functional) fragment" of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. The term "VL fragment” can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.

The term "VH fragment" (nanobody) can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

The term "Fd fragment" can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e., VH and CH-i. The "Fd fragment" does not include the light chain, or the second and third constant regions of the heavy chain. The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about no amino acids of both the heavy and light chains.

The term "Fab fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains.

The term "Fab' fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain. The antibody fragment may alternatively comprise a Fab'2 fragment comprising the hinge portion of an antibody. The term ”F(ab) fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab) fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

The term "single chain Fv ( scFv )" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

The term "bispecific antibody ( BsAb )" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

The term "CDR" can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e., the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.

The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

The mature conjugate subunit may be created by cleaving the double-stranded polynucleotide of the immature conjugate subunit with an enzyme. The enzyme may be a nuclease enzyme. Most preferably the enzyme is an exonuclease. Exonucleases are enzymes that cleave terminal 3’ and/or 5’ nucleotides from a polynucleotide, such as a blunt-ended polynucleotide, to create sticky ends. The enzyme may be a 5’ exonuclease or a 3’ exonuclease. Preferably the enzyme is a 5’ exonuclease, such as a T5 exonuclease.

A sticky end refers to a series of unpaired nucleotides in a double stranded oligonucleotide. Sticky ends are created by nucleases (e.g., endonucleases or exonucleases) creating a staggered cut in a double-stranded polynucleotide. Nucleases typically have recognition sequences that vary in the number of nucleotides from 4 to 6 and even 8. The longer the sticky ends, the higher the melting temperature of the double-stranded polynucleotide and the higher the number of potential sequences for recognition. Also, the longer the sticky ends, the greater the possibility of a single-strand break (DNA base hydrolysis). Thus, the sticky ends of the nucleotides referred to herein may be at least about 5, at least or about 6 or at least or about 7 nucleotides in length. Preferably the sticky ends of the nucleotides are about 15 to about 25 nucleotides in length. Most preferably the sticky ends of the nucleotides are about 20 nucleotides in length. Sticky ends are preferably 20 nucleotides in length because it provides 160,000 potential sequences for recognition. Sticky ends that are about 20 nucleotides in length are also preferred because they provide a moderately high melting temperature, thus preventing separation of the strands during enzymatic construction of the modular polynucleotide.

The length of the sticky ends is dependent on the nuclease used to cleave the polynucleotide. The nuclease may be an exonuclease, preferably a T5 exonuclease. T5 exonucleases create sticky ends that are about 20 nucleotides in length.

The skilled person will appreciate that the enzyme used to create sticky ends in a double-stranded polynucleotide can also be used to cleave a separate double-stranded polynucleotide so as to make the sticky ends of both polynucleotides complementary. Thus, for example, the double-stranded polynucleotide of an immature conjugate subunit and a double-stranded polynucleotide spacer may be cleaved by the same nuclease (such as an exonuclease, e.g., T5 exonuclease) so as to create complementary sticky ends that may be used to anneal the spacer and subunit together to form a modular polynucleotide. Consequently, the modular polynucleotide may be created by one-pot synthesis (mixing all of the reactants in a single reaction vessel as opposed to making the modular polynucleotide in a stepwise fashion). The skilled person will also appreciate that one-pot synthesis can be used to create a modular polynucleotide according to the invention due to the polynucleotide of each immature conjugate subunit comprising a nucleotide sequence that once cleaved will be complementary to a separate mature conjugate subunit. Thus, once each polynucleotide (i.e., immature conjugate subunit and double-stranded polynucleotide spacer) in the one-pot synthesis reaction has been cleaved by a single nuclease (e.g., T5 exonuclease), only the polynucleotides with complementary sticky ends will anneal together. Thus, the order of the probes in a modular polynucleotide may be arranged at user’s discretion due to the modular nature of the mature conjugate subunits. The sticky ends referred to herein may be 5’ sticky ends or 3’ sticky ends.

The spacer may be a double-stranded polynucleotide. The spacer referred to herein may be a double-stranded polynucleotide comprising a nucleotide sequence that once cleaved (e.g., with an exonuclease, such as T5 exonuclease) will comprise one or two sticky ends that are complementary to the sticky ends of a mature conjugate subunit or will be complementary to the sticky ends of a mature conjugate subunit once it has been formed.

The spacer may comprise artificial nucleotides or natural polynucleotides. In one embodiment, the double-stranded polynucleotide is DNA. One or more of the nucleotides may comprise an epigenetic modification, such as a methylation.

The double-stranded polynucleotide spacer (e.g., DNA) may be at least about 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more base pairs in length. The double-stranded polynucleotide spacer (e.g., DNA) may be between about 300 and 2000 base pairs in length or between about 500 base pairs and about 1000 base pairs in length.

The presence of the spacer within the modular polynucleotide enables the distance between each probe to be controlled so as to increase the resolution of each signal produced by each probe but also to prevent each probe from being too spaced apart. Furthermore, the spacer can be used to control the order of the probes in the modular polynucleotide according to the invention.

The annealing step may comprise using any method known in the art to anneal complementary sticky ends together. The annealing step may comprise contacting a first mature conjugate subunit with a second or several other mature conjugate subunits. The annealing step may comprise contacting a mature conjugate subunit with a double-stranded polynucleotide spacer. The annealing step may comprise an enzyme. Preferably the annealing step occurs in the presence of an enzyme. The enzyme may be a DNA ligase and/or a DNA polymerase. DNA ligase joins or catalyses the joining of complementary sticky ends together. DNA polymerase inserts nucleotides into gaps of an incompletely synthesised/annealed double-stranded polynucleotide, such as modular polynucleotide referred to herein.

Preferably contacting a mature conjugate subunit with a double-stranded polynucleotide spacer occurs in the presence of a DNA ligase and a DNA polymerase. The DNA ligase may be Taq ligase. The DNA polymerase may be Taq DNA polymerase. Contacting a mature conjugate subunit with a double-stranded polynucleotide spacer may occur in the presence of a DNA ligase and a DNA polymerase for about 1 hour to 18 hours at about 40°C to about 50°C. Performing the annealing step under these conditions ensures that only complementary sticky ends anneal to each other (i.e., prevents mismatching of sticky ends). Mismatched base pairs are unstable at temperatures between about 40°C to about 50°C.

The modular polynucleotide may comprise two or more different probes that are capable of binding to the same analyte or a different analyte. The modular polynucleotide may comprise two or more different classes of probe that are capable of binding to the same analyte or a different analyte. The modular polynucleotide may comprise two or more identical probes.

The modular polynucleotides and mature conjugate subunits of the invention may be used for a variety of purposes, including for example, disease diagnosis, the food industry (e.g., testing food and water quality), waste analysis (e.g., nuclear and industrial waste analysis), environmental analysis (e.g., soil and atmosphere aspirational analysis).

According to another aspect of the invention there is provided a method of making a modular polynucleotide, the method comprising: i) conjugating a (first) probe to a probe conjugation site of a (first) double- stranded polynucleotide to create a (first) immature conjugate subunit, wherein the probe is for sterically hindering an enzyme that is capable of cleaving the double-stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end and/or a second sticky end; ii) repeating (i) to create a total of two or more immature separate conjugate subunits; and iii) converting the immature conjugate subunits of (ii) into separate mature conjugate subunits by cleaving the double-stranded polynucleotide of the immature conjugate subunits to form a first sticky end, or a first sticky end and/or a second sticky end; and iv) joining the sticky end(s) of the mature conjugate subunits together to form a single modular polynucleotide.

The probe acts as a steric hindering agent that impedes the activity of nucleases (e.g., exonucleases) that may be used to cleave short (i.e., less than 100 base pairs) blunt- ended double-stranded polynucleotides (e.g., an immature conjugate subunit). The inventors believe that the conjugation of the steric hindering agent to the conjugation site prevents that exonuclease from continuing its cleavage activity along the entire length of the polynucleotide comprising the sticky end(s). Consequently, the method according to the invention enables the blunt ends of a short double-stranded polynucleotide to be converted into “sticky ends” without converting the entire double-stranded polynucleotide into a single-stranded polynucleotide.

Thus, unlike the prior art methods, such as the Gibson assembly, the method according to the invention can be used to attach several different short, blunt-ended polynucleotides together. The blunt-ended polynucleotides may be between about 20 and about 100 base pairs in length or between about 34 and about 100 base pairs in length. In other words, the polynucleotide of the double-stranded immature conjugate subunit may be between about 20 and about 100 base pairs/nucleotides in length or between about 34 and about 100 base pairs/nucleotides in length.

The conjugating step may be performed using any technique referred to herein. The probe may be for binding an analyte. The probe may be any probe referred to herein.

Step (i) may be repeated to create 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, or 500 or more separate immature conjugate subunits. Step (i) may be repeated to create 3 (3mer) or more, 4 (4mer) or more, 5 (5mer) or more, 6 (6mer) or more, 7 (7mer) or more, 8 (8mer) or more, 9 (9mer) or more, 10 (lOmer) or more, 20 (20mer) or more, 50 (50mer) or more, 100 (lOOmer) or more, 200 (200mer) or more, or 500 (500mer) or more separate immature conjugate subunits. The combined total concentration of the immature conjugate subunits and any optional spacers used to create the modular polynucleotide may be about 0.5 pM to about 1 pM.

An enzyme (e.g., a 5’ exonuclease) may be used to cleave the double-stranded polynucleotide of the immature conjugate subunits so that they form sticky end(s). The enzyme may be a nuclease referred to herein. For example, the nuclease may be an exonuclease, such as a 5’ exonuclease. The 5’ exonuclease may be T5 exonuclease.

An enzyme may be used to join the sticky end(s) of the mature conjugate subunits together so that they form a modular polynucleotide. The enzyme may be a DNA ligase. The enzyme may be any DNA ligase referred to herein. Two or more enzymes (e.g., a DNA ligase and a DNA polymerase) may be used to join the sticky end(s) of the mature conjugate subunits together. The two enzymes may be DNA ligase and DNA polymerase. The DNA polymerase may be any DNA polymerase referred to herein.

The cleavage step and/or joining step may be performed at about 30°C to about 49°C or about 40°C to about 45°C. Preferably the cleavage step and the joining step are performed at about 40°C to about 45°C. At temperatures of about 50°C or greater and about 30°C or lower, the method forms little or no modular polynucleotides. The method may further comprise incubating the immature conjugate subunits together to so that join to form a modular polynucleotide. The incubation period may be about 90 minutes to about 16 hours.

In one embodiment, the cleavage step and the joining step are performed at about 40°C to about 45°C, and joining step comprises an incubation period of at least about 90 minutes.

In another embodiment, the cleavage step and the joining step are performed at about 40°C to about 45°C, and joining step comprises an incubation period of about 90 minutes to about 16 hours.

The method according to the invention may be an isothermal reaction. The isothermal reaction may be performed at about 40°C to about 45°C. The isothermal reaction may comprise an incubation period of about 90 minutes to about 16 hours. The isothermal reaction may be performed in the presence of a DNA ligase, a DNA polymerase, and an enzyme that is capable of cleaving the double-stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end, or a first sticky end and/or a second sticky end, such as a 5’ exonuclease. The isothermal reaction may be a one- step reaction. The isothermal reaction may be a two-step reaction. The isothermal reaction may be the Gibson Assembly.

The probe conjugation site may comprise a nucleotide sequence specific for a transferase enzyme. The nucleotide sequence specific for a transferase enzyme may comprise a CpG island or a “TCGA” nucleotide sequence. The transferase enzyme may be a methyl transferase.

All of the embodiments of this aspect may be combined with any of the aspects or embodiments herein in any combination, unless stated otherwise with reference to a specific combination.

The inventors have developed a kit comprising components that can be used with a carrier-enhanced resistive pulse technology (e.g., nanopore-based resistive pulse sensing or nanopipette-based resistive pulse sensing technology) to determine if one or more analytes is present in a sample.

According to a fourth aspect there is provided a kit for determining if one or more analyte(s) is/are present in a test sample, the kit comprising:

• a double-stranded polynucleotide spacer; and

• an (im)mature conjugate subunit according to the invention or an (im)mature conjugate subunit made by the method according to the invention; or

• a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention.

In one embodiment, there is provided a kit for diagnosing a test subject suffering from a medical condition, the kit comprising:

• a double-stranded polynucleotide spacer; and

• an (im)mature conjugate subunit according to the invention or an (im)mature conjugate subunit made by the method according to the invention; or • a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention, wherein the presence of one or more analyte(s) in a bodily sample from a test subject is indicative that the subject suffers from the medical condition, or wherein the absence of the one or more analyte(s) from a bodily sample from a test subject is indicative that the subject suffers from the medical condition.

A kit according to the fourth aspect may be used to make a modular polynucleotide according to the invention. A modular polynucleotide according to the invention may be used to perform multiplex, high-throughput analysis using a carrier-enhanced resistive pulse sensing technology for the detection of different analytes using different probes.

Preferably, the kit further comprises at least one control or reference sample. The kit may comprise a control (e.g., a negative control and/or a positive control). The negative control may be a sample that does not comprise one or more of the analyte(s) to be detected. The positive control may be a sample that comprises one or more of the analyte(s) to be detected. The kit may comprise a buffer for the samples. The kit may comprise one or more enzymes selected from the group consisting of a DNA polymerase, a nuclease (e.g., exonuclease) and a DNA ligase.

The fifth aspect provides a method of determining if one or more analyte(s) is/are present in a test sample, the method comprising: i. contacting a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention with a test sample; and then ii. analysing the modular polynucleotide using a carrier enhanced-resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the test sample.

The test sample may be an environmental sample (e.g., a soil sample or atmospheric sample), a food industry, a water sample, a waste sample (e.g., nuclear or industrial waste sample) or a bodily sample that has been taken from a test subject. Thus, the method according to the invention may not comprise taking a sample from a test subject or performing surgery on a test subject. The method according to the fifth aspect may be used to diagnose if a test subject suffers from a medical condition or disease.

Thus, the sixth aspect provides a method of diagnosing a test subject with a medical condition, the method comprising: i. contacting a modular polynucleotide according to the invention or a modular polynucleotide made by a method according to the invention with a bodily sample taken from the test subject; and then ii. analysing the modular polynucleotide using a carrier enhanced resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the bodily sample, wherein the presence in the sample of the one or more analyte(s) is/are indicative that the subject has the medical condition, or wherein the absence from the sample of the one or more analyte(s) is/are indicative that the subject has the medical condition.

The method according to the sixth aspect is advantageous because it can be used to detect several different types of biomarkers simultaneously. Thus, the method provides a faster way to detect several different analytes and can be used to provide a more accurate diagnosis than known methods.

The method of the sixth aspect may comprise administering a therapeutic agent that treats the medical condition or disease to a subject.

Therefore, in a seventh aspect, there is provided a method of treating a subject suffering from a medical condition, the method comprising: diagnosing a test subject with a medical condition using a method according to the invention; and administering a therapeutically effective amount of a therapeutic agent for treating the medical condition.

In another aspect, there is provided a therapeutic agent for use in treating a medical condition in a subject diagnosed with a medical condition using a method according to the invention. In an eighth aspect, there is provided a method of determining the efficacy of a therapeutic agent being used to treat a subject’s medical condition, the method comprising: i. diagnosing a test subject with a medical condition using a method according to the invention; ii. administering a therapeutically effective amount of a therapeutic agent for treating the medical condition; iii. contacting a modular polynucleotide- according to the invention or a modular polynucleotide made by a method according to the invention with a test sample; and iv. analysing the modular polynucleotide using a carrier-enhanced resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the test sample, wherein the presence or absence of the one or more analyte(s) in the test sample is indicative of the efficacy of the therapeutic agent being used to treat the test subject’s medical condition.

Resistive pulse sensing technology requires two solutions to be separated by a narrow channel. A voltage is applied across the channel and (charged) molecules from one solution will move through the channel in the direction of the electric field. As they cross through the channel, the current passing between the electrodes will change. The change in current, and the duration of the change are directionally proportional to the widest diameter, and dimensions of the molecule passing through (see Figure 6). Examples of carrier-enhanced resistive pulse sensing technology include nanopore- based resistive pulse sensing, such as a biological nanopore (e.g., a Phi29 Connector channel), and nanopipette-based resistive pulse sensing technology.

The analyte is an agent that is capable of being bound by a probe. The analyte may be any substance present in a test sample (e.g., a biological or bodily sample or a non- biological sample or an environmental sample, a waste sample, a food sample or a water sample). Thus, the analyte may be a biological agent or a chemical agent.

The sample may be a fluid, such as a liquid or a gas. The sample may be a gas that has been condensed into a liquid. Preferably the sample is a liquid. Preferably the analyte is at least 2 nm in length. The sample may be a biological sample, such as a biological liquid. Thus, the analyte can be an analyte that is secreted from cells. Alternatively, the analyte can be an analyte that is present inside cells such that the analyte must be extracted from the cells before the invention can be carried out. Alternatively, the analyte can be an analyte that is present in a sample of fluid in which the biological organism is located.

Thus, the sample may be in vitro, in vivo or ex vivo. Thus, the invention may be carried out in vitro on a sample obtained from or extracted from a biological organism.

The biological organism or test subject may be a bacterium, a protista, a fungi, a plant or an animal. The biological organism may be a mammal, such as a human. Thus, the sample may be a bodily sample, such as a mammalian bodily sample, e.g., a human bodily sample. The sample typically comprises a biological fluid sample of the organism (e.g., a human). The biological fluid sample may be cerebrospinal fluid (CSF), urine, lymph, saliva, mucus or amniotic fluid.

The biological organism may be a commercially farmed animal, such as a fish, a horse, cattle, sheep or a pig; a pet, such as a cat or a dog; or a lab animal such as a mouse, a rat, a hamster or a guinea pig. The plant may be a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton. The plant may be a tree, such as Hymenoscyphus fraxineus.

The analyte may be an amino acid, a polypeptide, a carbohydrate (e.g., a polysaccharide or a disaccharide or a monosaccharide), lipids (fat), vitamin, a mineral, a metabolite or a polynucleotide. The polypeptide may be a protein. The protein can be an enzyme, antibody, complement protein (immune reaction), hormone, growth factor or growth regulatory protein, such as a cytokine, a structural protein (such as actin), a cellular receptor proteins (MHC), a transporter protein, glycosylated proteins. The protein may be a bacterial protein, a fungal protein, a viral protein, a plant protein, an animal protein, a protista protein or a parasite-derived protein.

The analyte may be a biomarker. The biomarker may be any biomarker known in the art that is capable of being bound by a probe. For example, the analyte may be interleukin-6, which is a marker of sepsis; procalcitoninin, which is a marker of a bacterial infection etc.

The sample may be a non-biological sample or a chemical sample. The non-biological sample is preferably a fluid sample, e.g., a liquid sample or gaseous sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

Thus, the invention may be carried out on a sample that is known to contain or suspected to contain the analyte. The invention may be carried out on a sample that contains one or more analytes whose identity is unknown. Alternatively, the invention may be carried out on a sample to confirm the identity and/or concentration of one or more analytes whose presence in the sample is known or expected.

According to another aspect of the invention, there is provided a method of tagging a polynucleotide with an azide group, the method comprising: contacting a polynucleotide comprising a TCGA target site with an azide donor in order to tag the target site of the polynucleotide with an azide group.

The polynucleotide may be a double-stranded polynucleotide referred to herein, or a single-stranded polynucleotide.

An azide donor is a substance comprising an azide group with the ability to freely donate the azide group without reacting to other groups on the recipient molecule. Thus, the azide donor may be RAdoHcy-8-Hy-PEG-N3, a modified SAM molecule containing an azide group or an azide modified nucleotide.

The contacting step may be performed in the presence of a catalyst, such as an enzyme. The enzyme may be a methyl transferase, preferably Taql methyl transferase (EC 2 1.1.72).

The contacting step may be performed in the presence of a methyl transferase for at least about 20 minutes, at least about 30 minutes, at least about 30 minutes. The contacting step may be performed in the presence of a methyl transferase (e.g., Taql methyl transferase) for between about 20 minutes and 24 hours. Preferably the contacting step is performed in the presence of a methyl transferase for between about 30 minutes and 2 hours, or between about 30 minutes and 90 minutes.

The contacting step may be performed in the presence of a methyl transferase (e.g., Taql methyl transferase) at about 34°C to about 55°C, preferably at about 45°C to about 55°C. Most preferably the contacting step is performed in the presence of a methyl transferase for about 30 minutes to about 90 minutes at about 50°C.

The term “one or more” can mean two or more, three or more, four or more, five or more, six more, seven or more, eight or more, nine or more, 10 or more, 15 or more, or 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more or 1000. The term “one or more” can alternatively mean “all” .

A conjugation site or a probe conjugation site may comprise “a nucleotide sequence specific for a transferase enzyme ” .

“A nucleotide sequence specific for a transferase enzyme ” can refer to a nucleotide sequence that comprises nucleotides that can be (specifically) modified by a transferase enzyme, e.g., a CpG island or a CpG thereof.

The tern “CpG island” may refer to a region of DNA that comprises a large number of CpG dinucleotide repeats. The region of DNA may be at least 200 nucleotides in length and have a CpG% of at least 50%.

The term a “ (functional) fragment” thereof can refer to an analyte-binding fragment.

The term “comprising” may refer to “consisting of” or “consisting essentially of” .

All of the embodiments and features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects or embodiments in any combination, unless stated otherwise with reference to a specific combinations, for example, combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show embodiments of the invention may be put into effect, reference will now be made, by way of example, to the accompanying drawings, in which: -

Figure 1 is a schematic overview of a method of making a modular double-stranded polynucleotide according to the invention (the method is also referred to herein as Sterically Controlled Nuclease Enhanced DNA Assembly (SCoNE DNA Assembly, Formally IADL));

Figure 2 is an agarose gel showing an N of 1 SCoNE. (1) Gene Ruler lkbp, (2) Gene Ruler low range, (3) 200bp DNA fragment (NoLimit, Thermo Scientific™), (4) 2kbp DNA fragment (NoLimit, Thermo Scientific™), (5) Negative control, (6) IADL lpM, (7) Positive assembly fragment, and (8) IADL 5pM;

Figure 3 is an agarose gel showing an N of 2 SCoNE experiment. (1) Gene Ruler lkb, (2) Gene Ruler low range, (3) 2kbp DNA fragment (NoLimit, Thermo Scientific™), (4) Short PI strand (self-assembly check), (5) IADL lpM, (6) Positive assembly fragment, (7) Negative control, and (8) IADL 5pM;

Figure 4 is an agarose gel showing an N of 3 and 4 SCoNE experiment. (1) Gene Ruler lkbp, (2) IADL lpM, (3) IADL 0.5pM, (4) IADL 0.25pM, (5) IADL 0.125pM, (6) Positive Control, (7) Negative control, and (8) 2kbp fragment (NoLimit, Thermo Scientific™);

Figure 5 shows a Gibson fragment assembly highlighting the probe attached fragments (p) and the spacer fragments (s) of dsDNA. It also illustrates that the strand will not circularise due to the break formation between probe fragment 1 and spacer fragment 10; and

Figure 6 is (A) an illustration of the DNA double helix backbone with adjoining capture probes for metabolites (probes A1 and A3 are aptamers; probes A2 and A5 are single-stranded DNA or RNA; and probe A4 is an antibody that binds a protein). (B) The experimental setup highlighting the differences in the signal obtained using a carrier-enhanced resistive pulse sensing technology when: (Bl) no probe is bound, (B2) the analyte is not bound to the probe, and (B3) the analyte is bound to the probe.

Figure 7 is a 1% agarose gel highlighting the ability to create decamer SCoNE structures (lanes 6 and 7). Lane 1 Gene Ruler lkbp (Thermo Scientific™), lane 2 2kbp fragment (NoLimit, Thermo Scientific™), lane 3 lOkbp fragment (NoLimit, Thermo Scientific™) , lane 4 whole l DNA (Thermo Scientific™), lane 5 PCR amplified construct, lane 6 decamer SCoNE lpM (N=l), lane 7 decamer SCoNE lpM (N=2), lane 8 negative control. We show our ability to generate decamer structures (lanes 6 and 7, 18.1).

Figure 8 shows a 1% agarose gel run at 75V for 45 minutes which shows that the inventors were are able to extract biotin labelled p strands (yellow box, left hand box) from the reaction mixture (red box, right hand box) with lane 1, gene ruler, lane 4 biotin extracted p strands, lane 6 reaction mixture prior to assembly.

Figure 9 shows a 1% agarose gel run at 75V for 45 minutes illustrating the inventor’s ability to assemble, and extract 3mer scone structures (yellow box, left hand box) from an assembled sample (red box, middle box). This also shows the need for an extraction method comparing to the starting reaction mixture (blue box, right hand box).

Figure 10 show respective intensity compared to biotin group positioning.

Figure 11 shows ELISA and newly adapted ELISA protocols. Panel A shows a normal ELISA using the 3mer SCoNE structure for protein isolation using streptavidin to bind reactive biotin groups. A1 shows SCoNE bound protein binding to primary IL6 antibody. A2 shows Secondary IL6 antibody binding to protein. A3 shows ABC binding to biotin labelled antibody. A4 shows ABC converting TMB buffer to coloured compound for measurement. Panel B shows an ELISA using the 3mer SCoNE structure, but removing the secondary antibody and measuring the protein concentration based on the biotin binding alone. Bl shows SCoNE bound protein binding to primary IL6 antibody. B2 shows Secondary IL6 antibody not added B3 shows ABC binding to biotin labelled SCoNE. B4 shows ABC converting TMB buffer to coloured compound for measurement. Panel C shows a modified ELISA using the 4mer SCoNE structure where the primary antibody binds procalcitonin and the secondary binds IL6. Cl shows SCoNE bound protein binding to primary procalcitonin antibody C2 shows Secondary IL6 antibody binding to IL6 protein. C3 shows ABC binding to biotin labelled IL6 antibody. C4 shows ABC converting TMB buffer to coloured compound for measurement. Panel D shows a similar experiment to C using the 4mer SCoNE structure where the structure has not been incubated with IL6, therefore the secondary antibody cannot bind and no signal should be observable. D1 shows SCoNE bound protein binding to primary procalcitonin antibody. D2 shows Secondary IL6 antibody added but cannot bind anything so is washed off. D3 shows ABC cannot bind as biotin sites are blocked by streptavidin. D4 shows ABC not present so cannot convert TMB to colour.

Figure 12 shows percentage of SCoNE structures retained against expected during ELISA analysis. “Antibody secondary” as referred to in the key of figure 12 corresponds to the left most bar of the graph. “Streptavidin linker only” as referred to in the key of figure 12 corresponds to the second bar from the left of the graph. “Separate protein linker IL6” as referred to in the key of figure 12 corresponds to the third bar from the left of the graph. “Separate protein linker IL6 run 2” as referred to in the key of figure 12 corresponds to the fourth bar from the left of the graph. “Separate protein linker Pro” as referred to in the key of figure 12 corresponds to the fifth bar from the left of the graph. “Separate protein linker no IL6” as referred to in the key of figure 12 corresponds to the sixth bar from the left of the graph.

Figure 13 shows structure of the 4mer with biotin at positions 1 and 2, bound human IL6 on an aptamer at position 3 and a blank Procalcitonin at position 4.

Figure 14 shows all 4mer translocation events at different biases (V).

Figure 15 shows an inverse relationship between bias and event duration. “-0.5” as referred to in the key of figure 15 corresponds to the left most data point on the graph, indicated by “x”. “- 0.6” as referred to in the key of figure 15 corresponds to the second data point from the left on the graph, indicated by “x”. “-0.7” as referred to in the key of figure 15 corresponds to the third data point from the left on the graph, indicated by “x”. “-0.8” as referred to in the key of figure 15 corresponds to the fourth data point from the left on the graph, indicated by “x”. The values referred to in the key of figure 15 correspond to the bias voltage applied. Accordingly, reference to “-0.5”, as referred to in the key of figure 15 refers to “-0.5V”. As would be clear to the skilled person, reference to “-0.6” in the key of figure 15 thus refers to “-0.6V” and so on. Figure 16 shows a comparison between SCoNE DNA average events at -0.6 and -0.7V (A and B) to bare DNA at the same biases (C and D). Figure 17 shows a comparison of sub events analysis (frequency count) conducted between 4mer SCoNE DNA and bare 4kbp DNA fragments. A comparison between sub event threshold (A) shows that increasing threshold value decreases the number of peaks observable (25-175pA threshold respectively). A 3D illustration of subevent position along the DNA backbone across a range of biases shows similar positioning of the subevents (B), most notably between -0.6 and -0.7V (C). The 2D representation re-enforced these conclusions (D). Comparison between 4mer

SCoNE DNA and bare 4kbp DNA fragments shows a lack of additional subevent peaks in the bare DNA samples represented in 3D and 2D views for direct analysis (E and F).

Figure 18 shows examples of SCoNE DNA current-time traces (A, E and I) for -0.5V, -0.6V, and -0.7V respectively, with single events highlighted for each bias (B-D, F-H, and J-L).

Figure 19 shows examples of bare 4kbp DNA current-time traces (A, E and I) for -0.5V, 0.6V, and -0.7V respectively, with single events highlighted for each bias (B-D, F-H, and J-L). Figure 20 shows a 1% agarose gel run at 75V for 45 minutes illustrating the ability to assemble different SCoNE structures, and that without modifications to the pDNA strands, assembly is not possible. Lane 1 Gene ruler lkbp, lane 2-4 blank, lane 5 Gibson assembly using unmodified pDNA, lane 6 dimer SCoNE assembly, lane 7 tetramer SCoNE assembly, lane 8 decamer SCoNE assembly. The formed structures are illustrated for lane 5-8 with red circles representing the biotin linker groups and the Y shapes representing the aptamers.

Figure 21 is an illustration of the calibration curve calculated from the lkbp GeneRuler ladder with an exponential fit line and 95% confidence intervals, and the resultant sDNA and SCoNE fragment size calculated from this fit.

Figure 22 is a comparison between the predicted and measured sizes of the SCoNE structures illustrating high similarity Examples

Example 1 - overview of a method of making a modular polynucleotide Figure 1 is a schematic overview of a method of making mature conjugate subunits, which in turn, can be used to create a modular polynucleotide (F) according to the invention. As shown in step (A), a single aminated probe is conjugated to DBCO to create a modified probe for further conjugation. (B) Short, blunt-ended DNA fragments (less than 100 bp in length) are azidated using a methyl transferase (m.Taql), and an azide modified SAM. (C) Independently, the modified probes and their respective azidated short, blunt-ended DNA fragments are combined together in a copper-free azide-alkyne reaction (a Click-iT™ reaction) to form of immature conjugate subunits. (D) The immature conjugate subunits are then combined into a single vial along with spacer DNA strands (at least 100 bp in length), T5 exonuclease, Taq DNA Figase, and Taq DNA polymerase. (E) In this step, the 5’ ends of the spacer DNA strands are digested by T5 exonuclease to create sticky ends. In addition, the 5’ ends of the immature conjugate subunits are digested by T5 exonuclease to create mature conjugate subunits (i.e., short, probe bound DNA fragments with sticky ends, which, in this case, are complementary to the sticky ends of the spacer DNA). The T5 exonuclease is unable to navigate across the site at which the probe is attached to the polynucleotide of the immature conjugate and therefore gets knocked off. The spacer DNA strands with sticky ends and the mature conjugate subunits are thus able to form bonds via their matching sticky ends. DNA polymerase fills in any gaps created during digestion and DNA ligase seals the scars, forming phosphodiester bonds, thus resulting in the formation of a modular polynucleotide (a SCoNE structure) shown in (F).

Example 2 - Protocol for creating a modular polynucleotide Materials and Methods Stock concentrations 50mM DBCO-NHS-ester a) lmg of DBCO-NHS-ester added to 1ml of DMSO (HPLC grade) to create a 2.5mM stock b) Take Imΐ of the 2.5mM added to 49 mΐ of nuclease free water to create a working stock of 50mM reating the modified probe a) Each probe should be modified in a separate PCR tube, the volumes provided can be amplified as necessary b) Pipette 18 mΐ of 50mM DBCO-NHS-ester into a PCR grade nuclease free vial c) Add 2m1 of prepared amine linked capture probe of interest (prepared as indicated by the manufacturer) d) Incubate at room temperature for a minimum of 2 hours-overnight e) Modified probes using ssDNA and aptamers are stable at 23°C for 5 daysreating modified probe strands (Azidation) a) Each probe strand should be modified in a separate PCR tube, the volumes provided can be amplified as necessary b) Set up the PCR tube with the following reactants and their respective volume, ensuring that the m.Taql enzyme is the final component to be added. Final volume is 20m1. This can be scaled up dependant on the requirements. c) Incubate reaction vessel at 40°C for 2 hours d) Pipette 0.5 mΐ of Protinase K (18mg/ml) into the vial e) Incubate at 50°C for 1 hour f) Allow to cool to room temperature for 20 minutes g) Perform PCR clean-up protocol (GenElute™ Sigma-Aldrich) a. Ensure elution is into 20m1 of either elution buffer or nuclease free water b. Concentration should be determined at this point (e.g., using Nanodrop) h) Can be stored at 4°C until required ull construction of Probe strands a) Each probe strand should be constructed in a separate PCR tube, the volumes provided can be amplified as necessary. b) Pipette 4pl of azidated probe strand into a PCR tube c) Pipette 2m1 of modified probe into the same tube d) Incubate at room temperature for a minimum 1.5 hours, can be left overnight. e) Can be stored at 4°C until required ssembly a) All components should be added into the same PCR vial for assembly b) Pipette equal concentrations of the reagents into a PCR tube to create the IADL-mastermix. The current set up uses the following volumes due to concentration: c) Ensure the IADL-mastermix is mixed well using a pipette d) In a new PCR vial pipette 10m1 of the IADL-mastermix e) Pipette 5m1 of nuclease free water and mix well f) Pipette 5m1 of Gibson assembly mastermix [mastermix comprises a T5 exonuclease, a progressive polymerase (such as Taq polymerase) and Taq ligase (New England BioLabs - Gibson Assembly ® Master Mix / Gibson Assembly ©Cloning Kit - NEB # E2611S/L, # E5510S], and mix well (half of recommended) g) Incubate at 40°C for 1.5 hours (can be incubated at this temperature overnight) h) Desired product has been isolated

5. Current Isolation technique a) Run the product on a 1% agarose gel at 80V for 80 minutes in IX TAE buffer a. The product can now be isolated by size comparison (at approximately 0.5cm from pipetting well site) b) Excise the DNA band from the gel using a sterile scalpel c) Perform Gel extraction (GenElute™ Gel Extraction Kit Sigma-Aldrich) a. Ensure elution is into 20m1 of either elution buffer or nuclease free water b. Final concentration can be determined now d) Product is available for use

Example 3 - Confirmation of mature conjugate synthesis

Figure 2 is an agarose gel showing an N of 1 SCoNE (Sterically Controlled Nuclease Enhanced DNA Assembly), i.e., the method used to of make a modular double- stranded polynucleotide according to the invention. The gel demonstrates that in lanes 6 and 8 there are bands above the 2kbp cut off (as indicated by the line using the gene ruler, lane 1, and the 2kbp fragment, lane 4) illustrating that it is possible to form a dimer (2 probe strands, 2 spacer strands) using the SCoNE technique. This is further shown by the absence of this band in lane 5 (negative control) highlighting that it is the conjugation that allows the formation of the larger structures. The lower band at lkbp is unreacted spacer DNA.

Figure 3 is an agarose gel showing an N of 2 SCoNE experiments. The gel demonstrates it is possible to generate a dimer (2 probe strands, 2 spacer strands) using the scone technique, as highlighted in lanes 5 and 8. The absence of a fragment above 2kbp in lane 7 highlights that it is the use of the conjugates which allows for these fragments to be formed. The 2kbp cut-off height is indicated by the line using the gene ruler, lane 1, and the 2kbp fragment, lane 3. The lower band at lkbp is unreacted spacer DNA.

Figure 4 is an agarose gel showing an N of 3 and 4 SCoNE experiments. Lanes 2 and 3 (N3 and N4 respectively) highlight the formation of the dimer (2 probe strands, 2 spacer strands). The absence of these fragments in the negative controls in lanes 4 and 5 (N3 and N4 respectively) highlight that it is the conjugation of the probes which allow for these fragments to be formed. The 2kbp cutoff height is indicated by the line using the gene ruler, lane 1, and the 2kbp fragment, lane 8. The lower band at lkbp is unreacted spacer DNA.

Figure 7 is an agarose gel showing several intermediate steps are generated during the reaction (lanes 6 and 7, 18.2 and 18.3), which indicates the depletion of the starting DNA. The absence of lkbp DNA (lanes 6 and 7, B.4) indicates that the spacer DNA has been fully incorporated into SCoNE structures unlike in the negative control (lane 8, B.5) where the lkbp fragments are still present.

Example 4 - Isolation of SCoNE

As described above, the inventors have shown that they are able to create structures with the expected weight. This was shown in 1% agarose gels.

This example relates to isolation. The nomenclature provided in the table immediately below is relevant. In this example, experiments were performed to increase yield of single product collection, removing unwanted DNA fragments from the initial one pot reaction mixture, and to determine the most effective positioning of the biotin groups to allow for this.

Nomenclature Explanation

S Spacer DNA strand (lOOObp)

P Probe DNA strand with aptamer attached (60bp) B Probe DNA strand with biotin group attached (60bp)

3mer A SCoNE structure comprised of 3S and 3P or B strands

4mer A SCoNE structure comprised of 4S and 4P or B strands 3S,1P3, 2B1,2 Indicates 3 S strands, 1 P strand at the third position, and 2 B strands at positions 1 and 2

The inventors have included an additional probe structure (DBCO-dPEG® 12-biotin, Sigma-Aldrich) to assist with isolation. The biotin groups added to the SCoNE structure were tested for their position effectiveness and the effectiveness of their use as a purification method. The inventors found that positions 1 and 2 of the final structure provided the highest yield. Isolation method

First, the inventors directly tested their ability to create the new biotin structures and tested their isolation method for these. Lane gaps between samples and the ruler were added to increase resolution of the low concentration samples as shown in figure 8. From these results the inventors were able to show they can create p strands containing biotin and extract them from starting materials prior to the assembly step.

The positioning of the biotin group within the SCoNE structure was then compared with the achieved yield. Due to working in very low concentrations it was difficult to use gel electrophoresis for complete quantitation, however Gray values were compared across all groups tested as shown in figure 9.

From these results, the inventors were able to show that they can extract assembled SCoNE structures using the biotin tag they added in from the mix of starting elements. As is shown in figure 10, the inventors also demonstrated that the positioning of the biotin group is also important for extraction.

This difference shows that biotin at positions 1 and 2 provide the highest efficiency in extraction. This could be due to multiple bindings to streptavidin during extraction assisting the binding of the SCoNE structure. The inventors observed a decrease in binding of 2.60%, 2.88%, 1.21%, and 2.48% respective to 3S,1P3,2B1,2.

Testing binding capacity

To perform binding capacities and efficiency of final structure formation, one standard (A) and three custom designed (B-D) ELISAs were performed, illustrated in figure 11. Several wash steps were performed between steps.

Notes on extraction and utilisation of SCoNE structures

Scone structures are generated at different concentrations dependant on the vial used, therefore data is normalised to expected SCoNE concentration. Nanodrop is used to determine starting SCoNE concentration. Through calculating yields of SCoNE structures through the gel experiments, 3mer and 4mer structures were generated with efficiencies of 57-61%. Some of the SCoNE structures are also lost during extraction, this loss is approximately 20%. As the inventors extract twice, this is taken into account during calculations. Where applicable to bind the biotin groups on the SCoNE structure, an excess of 300%, to the expected concentration, streptavidin was used and incubated for 30 minutes prior to experiments taking place. SCoNE structures were incubated for 30 minutes with the respective protein/s at 0.5ng/ml. SCoNE structures from different assembly reactions were used for each experiment to ensure reliability in the assembly and isolation protocols. All experiments were completed to N=6. Expected concentrations were 54.45, 54.49, 21.50, and 13.82 ng/ml (for IL6 run 2, procalcitonin, and no IL6 experiments).

Results and discussion

From the results of the ELISA performed, the inventors showed that they were able to successfully isolate SCoNE structures with functional capture groups. Results from the ELISAs are split into the 3mer and 4mer experiments. With respect to the starting concentrations expected, the yield of producing and successfully binding the proteins of interest are 71.31% (ELISA A), 67.49% (ELISA B), 54.00% (ELISA C), 74.88% (ELISA C), and 65.91% (ELISA C, using the reverse antibody set; IL6 primary with procalcitonin secondary). When removing the IL6 protein for ELISA D, a yield of 1.80% was observed. This indicates that not all biotin sites were fully occupied, therefore an error of 1.8% can be applied to all values obtained, bar ELISA B experiments.

Example 5 -translocation of SCoNE through a nanopore

This example relates to translocation. Translocation of bound SCoNE DNA through a nanopore was performed to assess firstly, the ability of the sensing apparatus to accurately detect DNA translocation. Secondly, to categorise the profile of SCoNE DNA and its differences from that of bare DNA. Finally, inclusion of a bound probe was utilised to determine the stability of the probe binding, translocation potential under real experimental conditions, and develop data analysis tools for subsequent comparison. The inventors successfully detected SCoNE DNA with bound analyte and successfully determined its differences to that of bare DNA.

Methods

Cleaned amber liquid cells were filled with 2ml of the 4 M LiCl 10% TE solution. SCoNE DNA was added into the vial to achieve a final concentration of approximately 80pM. A size determined nanopipette was inserted into the cell, submerging the tip in the liquid. Anodized silver/ silver chloride electrodes, soldered to gold contact pins, were added to the setup, such that one electrode sat inside the pipette chamber, and the other in the bulk solution, outside of the pipette. This was then attached to a custom low noise amplifier, sampling at 1MHz. A 100kHz in- line filter was attached to the output of the amplifier, and connected directly to a Picoscope 4262 oscilloscope, which was used for real -time monitoring of the system. A custom MATLAB script was used to control the bias voltage applied to the system, which allowed for changing the input voltage during measurements. Each scan was saved for further event and sub event analysis performed by custom MATLAB scripts.

Translocation results

When translocating the 4mer structure, as indicated in figure 13, the inventors observed translocation of the 4mer structure as illustrated in figure 14. The inventors also showed that translocation follows the expected trend (to bare DNA) with increasing bias directly correlated with a decrease in event duration, figure 15. When comparing average translocation profiles, see figure 16, the inventors observed that whilst the SCoNE translocation events have significant current decreases at specific points along the translocation, the bare DNA has less structure to where sub events can occur. It was also observed that decreases in current are matched by similar returns to the positive in bare DNA, these positive spikes are not observed in SCoNE DNA. This is further highlighted when looking at singular events.

Sub event analysis provides a further insight into the substructure of the events as highlighted in figure 17. The inventors applied a threshold for determining sub event analysis of between 25-175pA (10. A). The DNA structure is approximately 4.1kbp long, and comparisons are made between SCoNE 4mer and 4kbp DNA fragments (No Limits). DNA has the capability to translocate both forwards and backwards through the nanopore. In several of the figures herein, the size of the backbone has been normalised to values 0—1 with the relative positions of sub structures falling between these values. In a forwards translocation, the inventors expected to see peaks at positions near 0, 0.25, 0.5 and 0.75. In a backwards translocation, the inventors expected to see peaks at positions 0.25, 0.5, 0.75 and near 1. As can be seen from the from figure 17A, there are several peaks which emerge from the events generated. In lower thresholds (25-75pA) the inventors observed peaks at near 0, near 0.25, 0.5, near 0.75 and some emergence near 1. Due to the size of the biotin binding groups, and the unbound aptamer, it is possible that during data acquisition some of the resolution near the beginning and end of the event is lost. When the inventors applied a threshold between 100-150pA, the inventors observed loss of these initial, and ending peaks, however obtained a greater resolution of subevents at positions 0.25 and 0.5. Above 150pA threshold the inventors observed only the peak at 0.25 remaining, indicating they had filtered out useful subevents. When the inventors use a lOOpA and visualise the event across the different biases applied (see figure 17B) they observed that there are clear peaks across the datasets at positions 0.25, 0.5, and potentially near 1. This is even visible in the 0.5V bias which had a very limited number of translocations. These peaks are most notable in the -0.6 and -0.7V biases (See figure 17C, and 17D). When the inventors compared the subevent analysis to bare 4kbp DNA (nearest comparable size), they observed that the bare DNA creates a smooth shape across the event, lacking notable subevent peaks across the backbone (see figure 17E and 17F). Due to differences in starting concentration, frequency counts have been normalised against total counts for direct comparison.

Single Event View When the inventors viewed singular events, they observed the trends observed in both the average analysis and the subevent analysis. Figure 18 highlights a few events (excluding A, E and I) observed in the scans (A, E and I) across three biases used during experiments (- 0.5V, -0.6V and -0.7V respectively). These show a clear definition of sub events appearing from the baseline as opposed to noise contribution. This can be determined by comparison to bare 4kbp DNA translocation events (figure 19, excluding A, E and I). Experiments were conducted using nanopores of similar size (usually between 10-20 nm pores, depending on the size of the analyte), with the only difference being the DNA in solution. It can also be noted that translocation frequency is much higher in bare 4kbp, this is due to a higher starting concentration.

Summary

Overall, the inventors have:

• demonstrated that SCoNE DNA can be translocated and sub-events can be detected successfully; · demonstrated that SCoNE DNA-protein binding can be successfully achieved;

• developed a DNA extraction method and tested this successfully;

• developed new ELISA methods and tested these for protein binding with high efficiency; and

• compared SCoNE to bare 4kbp DNA and shown a clear distinction between event shapes.

Example 6 - DNA mobility analysis Summary

• In the Examples above, we confirm the structures generated are of the size we expect them to be when comparing them to the predicted size fragments.

• No significant differences observed between predicted and measured SCoNE structure sizes. • Summary of the optimisation conditions explored, highlighting the functionality of the method.

To determine the size of the DNA fragments, the distance migrated by each fragment was measured from the centre of the initial well to the centre of the DNA band (See Figure 20). The DNA ladder was used to create a calibration curve, applying an exponential fit with a 95% confidence band. The equation of the line was then used to estimate the size of the SCoNE structures and the sDNA fragment. The result of this analysis is highlighted in figure 21. From the results of this analysis, it was possible to estimate that the size of the fragments was 904±198bp for sDNA, 2005±176bp for the dimer structure, 4142±211bp for the tetramer structure, and 11128±524bp for the decamer structure.

A comparison plot between the predicted values and the measured values was then generated and the slope and significance, using a one-way ANOVA, calculated. From the results of the analysis, the slope was calculated to be 1.007 and a significance at 0.96. Both results indicate significant similarity between the predicted and measured values. This result signifies that the structures generated are of approximately the size predicted, further suggesting the successful assembly of SCoNE structures.

Example 7 - Protocol optimisation

As mentioned above, the method developed differs from previously defined protocols for the individual assembly reactions. Optimisation of the SCoNE assembly technique illustrated that the suggested protocols had a negative impact on the formation of the SCoNE structures. Therefore, a wide variety of protocol parameters were modified to improve the yield of the final product, as is summarised in table 1 (below). When using the sulfo-DBCO NHS ester for pDNA assembly, we hypothesise that the presence of the negatively charged sulfonate group inhibits interaction with the DNA, decreasing the conjugation efficiency. The use of high salt concentration solutions (e.g., equal to or greater than about 0.1M). Gibson assembly is recommended to be performed at 50°C, denaturing the 5’ exonuclease quickly. However, when used as part of this invention, it was found that a significantly lower yield was obtained when using the recommended parameters. This may be due to the separation of the short pDNA fragments, or the degradation of the capture probes leading to increased digestion by the 5’ exonuclease. By lowering the temperature of the reaction (e.g., 40°C to 45°C) and increasing the incubation duration (e.g., 1.5 hours to overnight) the activity time of the 5’ exonuclease may be increased and the pDNA fragments remain stable. The inclusion of the biotin linker in place of the aptamer was shown not to impact the assembly reaction and improved the isolation of SCoNE structures.

Table 1 - Optimisation parameters explored for the development of SCoNE methodology with outcomes confirmed by gel electrophoresis.

Claims

1. A method of making a modular polynucleotide, the method comprising: i) conjugating a (first) probe to a probe conjugation site of a (first) double- stranded polynucleotide to create a (first) immature conjugate subunit, wherein the probe is for sterically hindering an enzyme that is capable of cleaving the double-stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end and/or a second sticky end; ii) repeating (i) to create a total of two or more immature separate conjugate subunits; and iii) converting the immature conjugate subunits of (ii) into separate mature conjugate subunits by cleaving the double-stranded polynucleotide of the immature conjugate subunits to form a first sticky end, or a first sticky end and/or a second sticky end; and iv) joining the sticky end(s) of the mature conjugate subunits together to form a single modular polynucleotide.

2 The method of claim 1, wherein the probe is for binding an analyte. 3. The method of claim 1 or claim 2, wherein step (ii) comprises repeating (i) to create 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, or 500 or more separate immature conjugate subunits.

4. The method of any one of the preceding claims, wherein a 5’ exonuclease is used to cleave the double-stranded polynucleotide of the immature conjugate subunits so that they form sticky end(s).

5. The method of claim 4, wherein a DNA ligase and a DNA polymerase are used to join the sticky end(s) of the mature conjugate subunits together.

6. The method of any one of the preceding claims, wherein the method is an isothermal reaction.

7. The method of claim 6, wherein the isothermal reaction is performed at about 40°C to about 45°C. 8. The method of claim 6 or claim 7, wherein the isothermal reaction comprises an incubation period of about 90 minutes to about 16 hours. 9. The method of any one of the preceding claims, wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme, optionally a CpG island or a “TCGA” nucleotide sequence.

10 The method of any one of the preceding claims, wherein the polynucleotide of the double-stranded mature conjugate subunit is between about 20 and about 100 base pairs/nucleotides in length.

11 A method of making an immature conjugate subunit, the method comprising: conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit.

12. A method of making a mature conjugate subunit, the method comprising: conjugating a (first) probe, for binding a (first) analyte, to a probe conjugation site of a (first) double-stranded polynucleotide, to create a (first) immature conjugate subunit; and forming a (first) mature conjugate subunit by cleaving the double - stranded polynucleotide of the (first) immature conjugate subunit to form a first sticky end, or a first sticky end and/or a second sticky end.

13. The method according to claim 12, further comprising: annealing the first sticky end or the second sticky end of the (first) mature conjugate subunit to a complementary sticky end of a (first) double - stranded polynucleotide spacer.

14. The method according to any one of the preceding claims, wherein conjugating comprises contacting a polynucleotide having a probe conjugation site functionalised with an azide group with a probe functionalised with a DBCO- NHS ester.

15. The method according to any one of the preceding claims, wherein the polynucleotide of the immature conjugate subunit comprises a first blunt end and/or a second blunt end. 16. The method according to any one of claims 12 to 15, wherein the polynucleotide of the double-stranded mature conjugate subunit is between about 20 and about 100 base pairs/nucleotides in length.

17. The method according to any one of claims 12 to 16, wherein the double- stranded polynucleotide of the mature conjugate subunit comprises 5 ’-sticky ends and/or 3 ’-sticky ends.

18. The method according to any one of claims 4 and 12 to 17, wherein the mature conjugate subunit is formed by cleaving the double-stranded polynucleotide of the immature conjugate subunit with an enzyme, optionally wherein the enzyme is a 5’ exonuclease, such as a T5 exonuclease.

19. The method according to any one of claims 13 to 18, wherein annealing comprises contacting a (first) mature conjugate subunit with the (first) double- stranded polynucleotide spacer in the presence of a DNA ligase and a DNA polymerase. 0 A method of making a modular polynucleotide comprising:

1 creating a mature conjugate subunit according to the method of any one of claims 12 to 19;

11 repeating the method of any one of claims 12 to 19 to create a total of two or more mature conjugate subunits, wherein each of the two or more mature conjugate subunits has a (first or second) sticky end that is complementary with a (first or second) sticky end of a separate mature conjugate subunit; and iii. annealing the complementary sticky ends of the two or more mature conjugate subunits to create a modular polynucleotide comprising a double-stranded polynucleotide backbone having two or more mature conjugate subunits. 21 The method according to claim 20, wherein annealing comprises contacting the two or more mature conjugate subunits in the presence of a DNA ligase and a DNA polymerase. 22. The method according to claim 5, 19 or 21, wherein the DNA ligase is Taq ligase.

23. The method according to claim 5, 19, 21 or 22, wherein the DNA polymerase is Taq DNA polymerase.

24. An immature conjugate subunit comprising: a probe, for binding an analyte, conjugated to a probe conjugation site of a double-stranded polynucleotide, wherein the probe conjugation site comprises a nucleotide sequence specific for a transferase enzyme.

25. The immature conjugate subunit according to claim 24, wherein the polynucleotide of the double-stranded mature conjugate subunit is between about 20 and about 100 base pairs/nucleotides in length.

26. The immature conjugate subunit according to claim 14 or claim 25, wherein the polynucleotide of the double-stranded mature conjugate subunit comprises a first blunt end or a second blunt end, and/or wherein the polynucleotide of the double-stranded mature conjugate subunit comprises 5’-ticky ends or 3’- sticky ends.

27. A double-stranded modular polynucleotide comprising: a double-stranded polynucleotide backbone comprising a plurality of probe conjugation sites, each site having a nucleotide sequence specific for a transferase enzyme, wherein each probe conjugation sites is separated by at least 16 base pairs, and wherein at least one probe conjugation site is conjugated to a single probe for binding an analyte.

28. The immature conjugate according to any one of claims 24 to 26 or the double- stranded modular polynucleotide according to claim 27, wherein the transferase enzyme is a methyl transferase, and/or wherein the nucleotide sequence specific for a transferase enzyme comprises a CpG island or a “TCGA” nucleotide sequence. 29. The double-stranded modular polynucleotide according to claim 27 or claim

28, wherein each of the probe conjugation sites is separated by at least about 30 base pairs.

30. The method of any one of claims 12 to 23, or the immature conjugate subunit according to any one of claims 24 to 26, or the double-stranded modular polynucleotide according to any one of claims 27 to 29, wherein the probe(s) is/are one or more selected from the group consisting of a single-stranded nucleotide, an antibody, a (functional) fragment of an antibody and/or an aptamer.

31. A kit for determining if one or more analyte(s) is/are present in a sample, the kit comprising: i. a double-stranded polynucleotide spacer; and ii. an immature conjugate subunit according to any one of claims 24 to 26 or an immature conjugate subunit made by the method of claim 11; or iii. a modular polynucleotide according to any one of claims 27 to 30 or made by the method of any one of claims 1 to 10 or 20 to 23.

32. A kit for diagnosing a test subject suffering from a medical condition, the kit comprising: i. a double-stranded polynucleotide spacer; and ii. an immature conjugate subunit according to any one of claims 24 to 26 or an immature conjugate subunit made by the method of claim 1; or iii. a modular polynucleotide according to any one of claims 27 to 30, wherein the presence of one or more analyte(s) in a bodily sample from a test subject is indicative that the subject suffers from the medical condition, or wherein the absence of the one or more analyte(s) from a bodily sample from a test subject is indicative that the subject suffers from the medical condition.

33. The kit of claim 31 or 32, wherein the kit comprises a negative control and/or a positive control.

34. The kit of any one of claims 31 to 33, wherein the kit comprises a buffer and/or one or more enzymes selected from the group consisting of a DNA polymerase, a nuclease (e.g., exonuclease) and a DNA ligase.

35. A method of determining if one or more analyte(s) is/are present in a sample, the method comprising: contacting a modular polynucleotide according to any one of claims 27 to 30, or produced by a method according to any one of claims 1 to 10 or 20 to 23, with a test sample; and then

11 analysing the modular polynucleotide using a carrier enhanced-resistance pulse sensing technology to determine if one or more analyte(s) is/are present in the test sample.