METHODS FOR ISOLATING DNA FROM A BLOOD SAMPLE

Abstract

The present invention relates to methods for isolating nucleic acids present in a sample, in particular cell-free DNA (cfDNA) from a blood sample and polymers, substrates and kits for the method. Polymers with characteristics suitable to bind such nucleic acids are provided.

Claims

1. A nucleobase containing polymer comprising a polymer backbone with at least a portion of side chains being nucleobase side chains, wherein the polymer further comprises positively charged moieties at physiological pH adapted to electrostatically interact with nucleic acids, and/or the polymer is adapted to interact with nucleic acids via hydrogen bonding.

2. The nucleobase containing polymer according to claim 1, comprising a polymer backbone with at least a portion of side chains being nucleobase side chains selected from one or more of the following: adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof or combinations thereof.

3. The nucleobase containing polymer according to claim 2, wherein the proportion of side chains of the polymer comprising the nucleobase is at least 1% but not more than 17%.

4. The nucleobase containing polymer according to claim 1, wherein the polymer comprises one or more monomers selected from the group consisting of 2-methoxyethyl acrylate (MEA), 2-methoxyethyl methacrylate (MEMA), diethylamino ethylacrylate (DEAEA), diethylamino ethyl methacrylate (DEAEMA), hydroxyethyl acrylate, hydroxyethyl methacrylate, methyl methacrylate, methyl acrylate, styrene, methyl styrene, glycidyl acrylate, glycidyl methacrylate, N-vinylacetamide, 2-methyl-2-nitropropyl methacrylate, acrylic acid, methacrylic acid, 2-[[(butylamino)carbonyl]oxy]ethylacrylate, dimethylamino ethyl acrylate, dimethylamino ethyl methacrylate, mono-2-(acryloyloxy)ethyl succinate, poly(ethylene glycol) methyl ether acrylate (PEGA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), thymine ethylacrylate (ThEA), thymine acetoxylethyl methacrylate (ThAcMA), combinations thereof, and derivatives thereof.

5. The nucleobase containing polymer according to claim 1, wherein the polymer comprises: (i) one or more monomers selected from: 2-methoxyethyl acrylate (MEA), 2-methoxyethyl methacrylate (MEMA), diethylamino ethylacrylate (DEAEA), diethylamino ethyl methacrylate (DEAEMA), hydroxyethyl acrylate, hydroxyethyl methacrylate, methyl methacrylate, methyl acrylate, styrene, methyl styrene, glycidyl acrylate, glycidyl methacrylate, N-vinylacetamide, 2-methyl-2-nitropropyl methacrylate, acrylic acid, methacrylic acid, 2-[[(butylamino)carbonyl]oxy]ethylacrylate, dimethylamino ethyl acrylate, dimethylamino ethyl methacrylate, mono-2-(acryloyloxy)ethyl succinate, poly(ethylene glycol) methyl ether acrylate (PEGA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), and combinations thereof; and (ii) one or more (meth)acrylate-based monomers with nucleobase side chains.

6. The nucleobase containing polymer according to claim 5, wherein the polymer comprises: (i) one or more (meth)acrylate-based monomers selected from the group consisting of MEA, MEMA, DEAEA, DEAEMA, PEGA and PEGMA; and (ii) one or more (meth)acrylate-based monomers with nucleobase side chains.

7. The nucleobase containing polymer according to claim 6, wherein the polymer consists of: (i) one or more (meth)acrylate-based monomers selected from the group consisting of MEA, MEMA, DEAEA, DEAEMA, PEGA and PEGMA; and (ii) one or more (meth)acrylate-based monomers with nucleobase side chains.

8. The nucleobase containing polymer according to claim 5, wherein the one or more (meth)acrylate-based monomers with nucleobase side chains are selected from thymine ethylacrylate, thymine acetoxylethyl methacrylate, combinations thereof, and derivatives thereof.

9. The nucleobase containing polymer according to claim 8, wherein the polymer comprises: (i) one or more (meth)acrylate-based monomers selected from the group consisting of: 2-methoxyethyl acrylate (MEA), 2-methoxyethyl methacrylate (MEMA), diethylamino ethyl acrylate(DEAEA), diethylamino ethyl methacrylate (DEAEMA), poly(ethylene glycol) methyl ether acrylate (PEGA), poly(ethylene glycol) methyl ether methacrylate (PEGMA) and combinations thereof, and (ii) one or more other (meth)acrylate-based monomers with nucleobase side chains selected from a group consisting of thymine ethyl acrylate (ThEA), thymine acetoxylethyl methacrylate (ThAcMA), and combinations thereof.

10. The nucleobase containing polymer according to claim 9, wherein the polymer comprises: about 10-50% 2-methoxyethyl acrylate, about 0-50% 2-methoxyethyl methacrylate, about 1-40% diethylamino ethyl acrylate, about 1-25% poly(ethylene glycol) methyl ether acrylate, and about 1-17% thymine ethyl acrylate and/or thymine acetoxylethyl methacrylate.

11. The nucleobase containing polymer according to claim 10, wherein the polymer comprises: about 30-50% 2-methoxyethyl acrylate, about 20-40% 2-methoxyethyl methacrylate, about 20-40% diethylamino ethyl acrylate, about 1-15% poly(ethylene glycol) methyl ether acrylate, and about 1-17% thymine ethyl acrylate and/or thymine acetoxylethyl methacrylate.

12. The nucleobase containing polymer according to claim 11, wherein the polymer comprises about 47% 2-methoxyethyl methacrylate; about 15% diethylamino ethyl acrylate, about 36% 2-methoxyethyl acrylate, about 1% poly(ethylene glycol) methyl ether acrylate, and about 1% thymine ethyl acrylate or thymine acetoxyethyl methacrylate.

13. The nucleobase containing polymer according to claim 9, wherein the polymer corresponds to one of the following structures: ##STR00004## wherein m is between 1 and 50, and n.sup.1, n.sup.2, n.sup.3, n.sup.4, and n.sup.5 are independently selected to provide a polymer with a composition as indicated in the following table: TABLE-US-00008 MEMA DEAEA MEA ThEA ThAcMA PEGA (%) (%) (%) (%) (%) (%) Ms13 39 32 29 Ms22 38 27 29 6 Ms23 37 26 28 9 Ms26 42 25 32 1 Ms27 38 26 28 8 Ms28 39 28 29 4 Ms29 33 28 25 14 MsY 39 30 29 1 1 MsX 47 15 36 1 1. wherein MEMA = 2-methoxyethyl methacrylate; DEAEA = diethylaminoethyl acrylate; MEA = 2- methoxyethyl acrylate; PEGA = poly(ethylene glycol) methyl ether acrylate; ThEA = thymine ethyl acrylate; and ThAcMA = thymine acetoxyethyl methacrylate.

14. The nucleobase containing polymer according to claim 1, comprising one or more (meth)acrylate-based monomers with nucleobase side chains and one or more other (meth)acrylate-based monomers.

15. The nucleobase containing polymer according to claim 14, wherein the one or more other (meth)arylate-based monomers are poly(ethylene glycol) methyl ether (meth)acrylates.

16. The nucleobase containing polymer according to claim 1, wherein the polymer comprises one or more diethylamine side chains which provides the polymer with a positive charge at physiological pH, and wherein said positive charge at physiological pH interacts electrostatically with nucleic acids.

17. The nucleobase containing polymer according to claim 16, wherein the backbone of the polymer is synthesized using at least 5% diethylaminoethylacrylate.

18. The nucleobase containing polymer according to claim 1, wherein the polymer has a molecular weight in the range of from 1,000 Da to 500,000 Da.

19. The nucleobase containing polymer according to claim 1, wherein the nucleic acid is cfDNA.

20. A method of synthesizing the nucleobase containing polymer according to claim 1, wherein said method is RAFT polymerization.

Description

Embodiments of the Present Invention Will Now be Described with Reference to the Accompanying Figures, by Way of Example Only, in which

[0110] FIG. 1: illustrates the synthesis of nucleobase containing polymers comprising thymine derivatives;

[0111] FIG. 2: illustrates substrates provided with the nucleobase containing polymers in which the polymers are provided on support mesh or sponge substrates to provide high surface areas for nucleic acid binding, the arrows noting a coating on the substrate;

[0112] FIG. 3: provides an overview of cfDNA isolation using the methods of the invention;

[0113] FIG. 4: illustrates the recovery of DNA (150 ng) from aqueous solution using a 125 mm.sup.3 polyurethane sponge coated with nucleobase containing polymer;

[0114] FIG. 5: illustrates the recovery of DNA (150 ng) from an aqueous solution blood mimic using a 125 mm.sup.3 polyurethane sponge coated with nucleobase containing polymer following 30 minutes of cycling the blood mimic at 28 mL/min at 37? C.;

[0115] FIG. 6: illustrates an overview of an extracorporeal extraction cfDNA isolation using the methods of the invention for cfDNA sampling;

[0116] FIG. 7: illustrates the sequestering of radiolabelled cfDNA from whole blood using a range of nucleobase containing polymers;

[0117] FIG. 8: illustrates the elution of cfDNA after scavenging from whole blood. The elution buffers used were EQ1 buffer=0.1 M sodium acetate pH 5, 0.6 M NaCl, 0.15% Triton X-100; W8 buffer=0.1 M sodium acetate pH 5, 0.825 M NaCl and E4 buffer=0.1 M Tris-HCl pH 8.5, 1.25 M NaCl.

[0118] FIG. 9: illustrates polymers (MS13=30% diethylaminoethylacrylate, MS22-28=30% diethylaminoethylacrylate and thymine monomer) based on DNA recovery yields after incubation with whole blood (human) spiked with DNA (mouse mononucleosomal). DNA was quantified by HS Qubit? and submitted for size analysis. Mouse mononucleosomal DNA (a model of cfDNA) was detected between 50-300 bp;

[0119] FIG. 10: provides details of polymer compositions; and

[0120] FIG. 11: illustrates Bioanalyzer traces (DNA fragment size analysis) and demonstrates successful recovery of small fragment DNA by the polymer-coated sponge but not by the sponge alone (A) Pure mononucleosomal DNA, (B) DNA recovered by polymer, and (C) DNA recovered by THE negative control.

[0121] FIG. 12: illustrates a number of polymers of the invention, where m=2-50.

[0122] FIG. 13: illustrates the haemocompatibility of a range of polymers of the invention. Polymer-coated sponges prepared using different concentrations of coating solutions were prepared, and a qualitative evaluation of coagulation was performed, where +=coagulation observable and ?=no evidence of coagulation.

[0123] FIG. 14: illustrates DNA recovery from polymers of the invention (msX) compared with THE sponges using a flow-based setup. TE buffer spiked with 200 ng mononucleosomal DNA (160 bp) was incubated with either 1, 2, or 3?125 mm.sup.3 sponges. Bound DNA was eluted and quantified by HS Qubit?. (n=3, error bars show standard deviation).

[0124] FIG. 15: illustrates the linear DNA binding capabilities of the msX-treated sponges of FIG. 14.

DETAILED DESCRIPTION

Example 1Synthesis of the Polymers of the Invention

[0125] FIG. 1 summarises the synthesis of a polymer of the invention; whereby a polymerisation reaction of acrylate/methacrylate monomers results in a nucleobase containing polymer. Monomers were combined in the presence of a chain transfer agent (2-Cyano-2-propyl dodecyl trithiocarbonate) and initiator (2,2-Azobis(2-methylpropionitrile)) at a typical ratio of 1000:5:1, or 1500:5:1, in DMSO. The reaction was degassed with N.sub.2 before heating to 60? C. for up to 7 days.

[0126] The reaction was stopped by cooling with dry ice and acetone, and exposed to air. The polymer was purified by precipitation first into water, then three times by dissolving in THE and precipitated in hexane. The polymer was then dried of solvent in a vacuum oven (40? C.).

Example 2Synthesis of Specific Polymers of the Invention

[0127] Polymers were typically synthesised on a 1 g scale. Monomers were passed through basic alumina columns to remove inhibitors. Purified monomers were combined with chain transfer agent (2-cyano-2-propyl dodecyl trithiocarbonate), and AIBN (5:1) in a microwave vial and dissolved in DMSO to a final reaction concentration of 5 M. Reactions were degassed with N.sub.2 for 30 min. Polymerisations were stirred at 60? C. under N.sub.2 for 48 h. Reactions were quenched by exposing to air and cooling with acetone: dry ice. Polymers were first precipitated into water, then dissolved and re-precipitated three times using THE and hexane, respectively. Polymers were then dried by vacuum oven (40? C.).

[0128] Proportions of monomers that were used in the synthesis of the polymers are provided in Table A

TABLE-US-00006 TABLE A MEMA DEAEA MEA ThEA ThAcMA PEGA (%) (%) (%) (%) (%) (%) Ms13 40 30 30 Ms22 38 28.5 28.5 5 Ms23 38 28.5 28.5 5 Ms26 39 30 30 1 Ms27 36 27 27 10 Ms28 39 30 30 1 Ms29 36 27 27 10 MsY 38.5 30 29.5 1 1 MsX 51 10 37 1 1 wherein MEMA = 2-methoxyethyl methacrylate, DEAEA = diethylamino ethyl acrylate, MEA = 2-methoxyethyl acrylate, ThEA = thymine ethyl acrylate, ThAcMA = thymine acetoxyethyl methacrylate, and PEGA = Poly(ethylene glycol) methyl ether acrylate, Mn ~480
.sup.1H-NMR was used to measure the composition of the respective polymers following wherein the results are provided in Table B. More specifically, NMR characterisation was undertaken using a process wherein 5 mg of dried polymer was dissolved in 600 ?L DMSO-d6 and submitted for compositional analysis using .sup.1H-NMR, recorded using a Bruker AVA-500 at 500 MHz and 298 K. The broad peak corresponding to 2H (3.80-4.30 ppm) on the carbon alpha to the carboxylate on polymer side chains was set to represent 100% of the monomers components. This peak is shared by all monomers incorporated in the polymer. Individual monomer components were identified in reference to pure monomer samples, integrated, and calculated as a proportion of all monomers components.

TABLE-US-00007 TABLE B MEMA DEAEA MEA ThEA ThAcMA PEGA (%) (%) (%) (%) (%) (%) Ms13 39 32 29 Ms22 38 27 29 6 Ms23 37 26 28 9 Ms26 42 25 32 1 Ms27 38 26 28 8 Ms28 39 28 29 4 Ms29 33 28 25 14 MsY 39 30 29 1 1 MsX 47 15 36 1 1 wherein MEMA = 2-methoxyethyl methacrylate DEAEA = diethylamino ethyl acrylate, MEA = 2-methoxyethyl acrylate, ThEA = thymine ethyl acrylate. ThAcMA = thymine acetoxyethyl methacrylate, and PEGA = Poly(ethylene glycol) methyl ether acrylate, Mn ~480

Example 3Coating of the Polymers onto a Substrate

[0129] A nucleobase containing polymer was dissolved in THE and coated onto a support substrate provided by a sponge. FIG. 2 demonstrates this as viewed by scanning electron microscopy (SEM). Topographic changes highlight that the polymer has formed a layer on the surfaces of the substrate.

Example 4Use of a Nucleobase Containing Polymer to Isolate DNA

[0130] An embodiment of the process of isolating DNA from a solution 110 is shown in FIG. 3, wherein a substrate comprising a polymer as discussed herein 120 would be provided in a solution of substantially undiluted whole blood 130, which comprises cfDNA. The cfDNA in the blood would then bind to the polymer. A wash buffer 140 would then be used to remove residual material from the whole blood while retaining the cfDNA on the polymer. The wash buffer in this embodiment would comprise phosphate buffered saline (PBS). An elution buffer 150 would then be provided to elute the cfDNA from the polymer. In this embodiment the elution buffer was 100 mM Tris-HCl pH 8.5, 1250 mM NaCl (high salt buffer with a NaCl concentration >600 mM). The elution may then be purified or concentrated using conventional means to produce an isolated cfDNA sample which may then be characterised.

[0131] In the present example, a 125 mm.sup.3 polyurethane sponge was used as a substrate onto which a nucleobase containing polymer described herein was provided. Mouse mononucleosomal DNA in Tris-EDTA (TE) buffer (a blood mimic) was provided to the substrate. Compared to a control sponge treated with tetrahydrofuran (THF), the polymer-treated sponge retained significantly more DNA, as shown in FIG. 4: quantified by HS Qubit?, Thermofisher?.

[0132] The isolation of DNA from a flowing blood mimic using an embodiment of the invention is shown in FIG. 5. TE buffer treated with mouse mononucleosomal DNA was passed through a polyurethane sponge coated with a nucleobase containing polymer of the invention. After 30 minutes of cycling the blood mimic through the device at 28 mL/min at 37? C., the sponge was washed with PBS and the DNA eluted with elution buffer as discussed above (high salt buffer (>600 mM NaCl)). Compared to a control sponge treated with THF, the polymer-treated sponge coated with ms22 bound and eluted markedly more DNA from solution: quantified by HS Qubit?, Thermofisher?.

Example 5Use of the Nucleobase Containing Polymers to Isolate DNA

[0133] FIG. 6 describes an extracorporeal device 210 application of a nucleobase containing polymer as described herein wherein the polymer is bound to a substrate 220. Whole blood 230 is removed from a subject 240 and provided to a system comprising a sampling loop 250 before being returned 260 to the subject. To the whole blood may be added an anticoagulant 270 to prevent blood clotting while it is extracorporeal. The sampling loop comprises the polymer bound to a substrate 220. The sampling loop further comprises a peristaltic pump 280, and bubble trap or traps 290. When whole blood passes through the substrate, cfDNA binds to the polymer and other whole blood components do not. When sufficient cfDNA has been bound to the polymer, the substrate is removed from the sampling loop 300. The substrate is suitably washed such that the cfDNA is retained while other blood components are removed from the substrate. The cfDNA is then eluted under suitable conditions to provide a concentrated sample of cfDNA for characterisation 310.

Example 6Identification of Polymer Compositions and Elution Conditions

[0134] As shown in FIG. 7, several different polymer compositions with various concentrations and types of nucleobase- and non-nucleobase-containing side chain were produced and screened for DNA recovery from whole blood (pig) spiked with DNA (.sup.3H-labelled chromatin: mouse mononucleosomal). Polyurethane sponges were used as substrates for the polymers, and each coated sponge was incubated in the spiked blood for 30 minutes at 37? C. The sponges were then removed from the spiked blood and washed with PBS before elution of the bound DNA from the polymers using an elution buffer consisting of 0.1 M sodium acetate, pH 5, 0.6 M NaCl, 0.15% Triton X-100 (EQ1 Buffer, Invitrogen?). The concentration of labelled DNA was quantified using liquid scintillation counting, and the comparative binding efficiencies of the polymers indicated in FIG. 10. Because the measured .sup.3H signal can be influenced by the composition of the measurement solution, the results are represented as proportions of the signal measured from the quantity of DNA spiked into blood, in EQ1.

[0135] FIG. 9 identifies ms26 and ms28 as preferred embodiments of the invention due to their high retention and elution of DNA. FIG. 10 indicates compositions of polymers.

[0136] Various elution buffers were tested for their ability to release DNA from the polymers of the invention. Polyurethane sponges were coated with polymer of the invention in THE and each sponge was incubated in the whole blood (porcine) for 30 minutes at 37? C. The sponges were then removed from the whole blood and washed with PBS before elution of the bound DNA from the polymers using various elution buffers: EQ1, W8 or E4 (W8: 0.1 M sodium acetate, pH 5, 0.825 M NaCl, E4 buffer: 0.1 Tris-HCl, pH 8.5, 1.25 M NaCl). The concentration of labelled DNA was quantified using HS Qubit?, after purification using QIAamp Circulating Nucleix Acid Kit, and the comparative elution efficiencies of the elution buffers indicated in FIG. 8. A negative control sponge treated with THE was included for comparison, as well as a plasma control wherein isolated plasma (and thus concentrated cfDNA) was isolated and purified using QIAamp Circulating Nucleic Acid Kit,?. The results indicate that compared to the QIAamp kit, a comparable recovery method was established when elution is performed using high salt buffer as described herein. Plasma control generated by spinning test volume of blood (1 mL) at 2000 rcf for 10 min. The plasma supernatant was removed and made up to 1 mL with PBS before running on the QIAamp kit.

[0137] FIG. 8 indicates that a preferred elution buffer is one with a high salt concentration, and/or one with a high pH, for example 100 mM Tris-HCl pH 8.5, 1250 mM NaCl (high salt buffer with a NaCl concentration >600 mM).

Example 7Binding of nucleic acids to polymers

[0138] To identify whether polymers bound to and allowed isolation of circulating cfDNA from whole blood (which are typically 50-300 bp) as opposed to large DNA molecules (such as chromosomal DNA or DNA from leukocytes), polymers of the invention were prepared and coated onto polyurethane sponge while varying the composition of the polymers. These were provided to whole blood (human) spiked with mouse mononucleosomal chromatin, DNA was isolated and purified from the whole blood for 30 min at 37? C. incubation, PBS wash, elution with 100 mM Tris-HCl pH 8.5, 1250 mM NaCl (high salt buffer with a NaCl concentration >600 mM) and the DNA concentration of the eluted samples quantified by HS Qubit? as shown in FIG. 9. The isolated DNA was then quantified by the addition of a fluorescent dye, DNA electrophoresis and optical densitometry using an Agilent? Bioanalyzer?.

[0139] FIG. 9 indicates an embodiment of the invention for isolating short (50-300 bp) lengths of nucleic acids:polymer designated ms26, which permitted not only a high recovery of DNA from whole blood (FIG. 9), but the DNA which it retained had the highest proportion of short DNA than any polymer tested.

[0140] The Bioanalyzer? results of ms26 are shown in FIG. 11: (top) pure mouse mononucleosomal DNA, (middle) recovered DNA from polymer sponge, including small fragment mouse mononucleosomal DNA, (bottom) an untreated sponge lacking the polymer does not recover small fragment DNA.

Example 8Haemocompatability Assay

[0141] It is beneficial that the polymers of the invention are haemocompatible (e.g. they cause minimal blood coagulation in use). This is particularly relevant for the use of the polymers of the invention as illustrated in FIG. 6. To that end, FIG. 13 indicates a qualitative coagulation assay using various polymers of the invention at different concentrations of coating solutions, where +=coagulation observable, and -=no evidence of coagulation.

[0142] FIG. 13 identifies msX as a preferred embodiment of the invention due to the improved haemocompatibility.

Example 9MsX DNA Binding Assay

[0143] To identify whether polymer MsX bound and allowed the isolation of DNA, 125 mm.sup.3 sponges treated with either MsX at 0.1% THE coating solution or THE coated, and their DNA binding ability was assayed using a flow-based setup. A solution of TE buffer spiked with 200 ng mononucleosomal DNA (160 bp) was incubated for 30 min at 37? C. with 1, 2 or 3 of the MsX or THF-treated sponges. These were then washed with PBS and bound DNA was eluted using 100 mM Tris-HCl pH 8.5, 1250 mM NaCl (high salt buffer with a NaCl concentration >600 mM). The DNA was quantified by HS Qubit? (n=3, error bars show standard deviation) as shown in FIG. 14. The amount of DNA bound by each MsX sponge as shown in FIG. 14 is compared in FIG. 15.

[0144] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.