Search

US-12624348-B2 - Enrichment method

US12624348B2US 12624348 B2US12624348 B2US 12624348B2US-12624348-B2

Abstract

A method is provided for enriching extracellular DNA from a biological sample comprising extracellular DNA and extracellular vesicles, wherein the method comprises: (a) preparing a binding mixture comprising—the biological sample, —a solid phase comprising anion exchange groups, —an acidic binding buffer comprising a buffering agent, and binding extracellular DNA to the solid phase comprising anion exchange groups; (b) separating the solid phase with the bound extracellular DNA from the remaining binding mixture, wherein the remaining binding mixture comprises extracellular vesicles. The method may furthermore comprise processing the remaining binding mixture to enrich one or more biological targets of interest therefrom, wherein processing may comprise (c) enriching as biological targets extracellular vesicles and/or extracellular RNA from the remaining binding mixture.

Inventors

  • Martin Schlumpberger
  • Karolin SPITZER

Assignees

  • QIAGEN GMBH

Dates

Publication Date
20260512
Application Date
20201216
Priority Date
20191216

Claims (20)

  1. 1 . A method for enriching extracellular DNA and sequentially extracellular vesicles and optionally extracellular RNA or for enriching extracellular vesicles and optionally extracellular RNA from a biological sample comprising extracellular DNA and extracellular vesicles, wherein the method comprises: (a) preparing a binding mixture comprising the biological sample, a solid phase comprising anion exchange groups, and an acidic binding buffer comprising a buffering agent, and binding extracellular DNA to the solid phase comprising anion exchange groups; (b) separating the solid phase with the bound extracellular DNA from the binding mixture to obtain a remaining binding mixture, wherein the remaining binding mixture comprises extracellular vesicles; and (c) processing the remaining binding mixture to enrich one or more biological targets of interest therefrom, wherein the processing comprises enriching extracellular vesicles and optionally extracellular RNA as biological targets of interest from the remaining binding mixture, wherein step (c) comprises enriching extracellular vesicles by binding to an anion exchange surface of a solid phase in the presence of a buffering agent, wherein the binding conditions used in step (a) for binding extracellular DNA differ from the binding conditions used in step (c) for binding extracellular vesicles and optionally extracellular RNA, and wherein the different binding conditions in step (a) and step (c) are achieved by at least one selected from the group consisting of: (i) the extracellular vesicle binding mixture of step (c) has a lower pH than the extracellular DNA binding mixture of step (a); (ii) the buffering agent introduced into the extracellular vesicle binding mixture in step (c) differs from the buffering agent in the extracellular DNA binding mixture in step (a) and (iii) the anion exchange surface of a solid phase in step (c) differs from the anion exchange groups of the solid phase in step (a).
  2. 2 . The method according to claim 1 wherein when enriching extracellular vesicles and optionally extracellular RNA from a biological sample comprising extracellular vesicles and extracellular DNA, in step (a) at least extracellular DNA is bound as non-target biomolecule to the solid phase.
  3. 3 . The method according to claim 1 , wherein the pH of the acidic binding buffer is in a range of 2.5 to 6.5.
  4. 4 . The method according to claim 1 , wherein in step (a) the pH of the binding mixture corresponds to the pH of the acidic binding buffer or deviates by ≤1.5 pH units from the pH of the acidic binding buffer.
  5. 5 . The method according to claim 1 , wherein in step (a) the pH of the binding mixture is in a range of 2.5 to 6.5.
  6. 6 . The method according to claim 1 , wherein in step (a) the pH of the binding mixture is ≥4, ≥4.2 or ≥4.5.
  7. 7 . The method according to claim 1 , wherein in step (a) the pH of the binding mixture is lower than the pKa of the ionized form of the anion exchange groups of the solid phase.
  8. 8 . The method according to claim 7 , wherein the pH is at least 1 unit lower than the pKa.
  9. 9 . The method according to claim 1 , wherein the acidic binding buffer of step (a) comprises a carboxylic acid based buffering agent.
  10. 10 . The method according to claim 1 , wherein the buffering agent comprises a carboxylic acid and a salt of said carboxylic acid.
  11. 11 . The method according to claim 6 , wherein the acidic binding buffer of step (a) has a pH of ≥3.5, and wherein the buffering agent comprises a buffer component selected from the group consisting of citrate, oxalate, formate, propionate, lactate and tartrate.
  12. 12 . The method according to claim 1 , wherein in step (a) the binding mixture comprises the buffering agent originating from the acidic binding buffer in a concentration selected from the group consisting of: (i) 0.5M or less; (ii) at least 15 mM; and (iii) the range of 15 mM to 250 mM.
  13. 13 . The method according to claim 1 , wherein the acidic binding buffer comprises a buffering salt as buffering agent and a non-buffering salt, and wherein the concentration of the non-buffering salt in the acidic binding buffer is 1M or less.
  14. 14 . The method according to claim 13 , wherein the non-buffering agent is an alkali metal salt.
  15. 15 . The method according to claim 13 , wherein the total salt concentration in the acidic binding buffer of step (a) is 1M or less.
  16. 16 . The method according to claim 1 , wherein the solid phase that is used in step (a) is particles.
  17. 17 . The method according to claim 1 , wherein the anion exchange groups comprise at least one primary, secondary or tertiary amino group.
  18. 18 . The method according to claim 17 , wherein the anion exchange group of the solid phase comprises one or more of primary, secondary and tertiary amines of the formula (R) 3 N, (R) 2 NH, RNH 2 and/or X—(CH 2 ) n —Y wherein X is (R) 2N, RNH or NH 2 , Y is (R) 2N, RNH or NH 2 , R is independently an optionally substituted linear, branched or cyclic alkyl, alkenyl, alkynyl or aryl substituent which optionally comprises one or more heteroatoms, and n is an integer in the range of from 0 to 20.
  19. 19 . The method according to claim 17 , wherein the anion exchange groups have at least one of the following characteristics: (i) they comprise at least one amino group, wherein the amino group is part of a heterocyclic or heteroaromatic ring; (ii) they comprise at least one amino group, wherein the amino group is part of an imidazole ring; (iii) they comprise histidine or histamine; (iv) they comprise at least one ionizable group having a pKa value of the ionized form within a range of 6 to about 13; or (v) they comprise any one or more of a trialkylamine group and/or a dialkylaminoalkyl group for extracellular cell-free DNA (cfDNA) binding; a trialkylamine group and/or a dialkylaminoalkyl group for cfDNA binding wherein the alkyl groups independently comprise 1-6, 1 to 5 or 1 to 4 carbon atoms; a trialkylamine group and/or a dialkylaminoalkyl group for cfDNA binding and no further ionizable groups for cfDNA binding; a silane group; a functionalization with trialkylsilanes; or a functionalization with N, N-dialkyl-3-aminoalkyl) trialkoxysilane, wherein the alkyl groups are selected from methyl-, ethyl-, propyl-, butyl-, or pentyl-groups.
  20. 20 . The method according to claim 16 , wherein the solid phase that is used in step (a) is magnetic particles, wherein the anion exchange groups of the magnetic particles comprise a trialkylamine group and wherein the acidic binding buffer has a pH of ≥3.5, and wherein the buffering agent comprises a buffer component selected from the group consisting of citrate, oxalate, formate, propionate, lactate and tartrate.

Description

FIELD OF THE DISCLOSURE The present invention pertains to methods for enriching (i) extracellular DNA and/or (ii) extracellular vesicles and/or extracellular RNA from a biological sample. BACKGROUND OF THE DISCLOSURE Extracellular nucleic acids from cell-free biofluids, such as plasma, serum, or urine, represent important analytes for diagnostics and research. Of particular relevance are extracellular nucleic acids, such as extracellular DNA (also referred to as “cell-free” or “cfDNA” herein) and extracellular RNA (also referred to as “cell-free” or “cfRNA” herein). Extracellular RNA is e.g. found in extracellular vesicles (EVs), which contain mRNA and miRNA. The extracellular RNA comprised in extracellular vesicles is also referred to as vesicular RNA. In addition, non-vesicular extracellular RNA exists, which is often associated with proteins (e.g. miRNAs associated with Ago2 proteins) and is thereby protected from degradation. The efficient capture of extracellular DNA, EVs and/or cfRNA from the same biological sample is challenging and prior art methods often use complex, time consuming workflows or expensive materials (WO 2012/087241, WO 2017/197399 and EP 2941629 B1). Moreover, currently available protocols do not allow to provide cfDNA and cfDNA and/or cfRNA comprising vesicular RNA in separate fractions. There is an increasing interest and need for further methods for enriching and thus isolating (i) extracellular DNA, (ii) EVs and/or extracellular RNA that comprises vesicular RNA. In particular, there is a need for improved methods, that are more simple than the prior art workflows and allow to provide (i) extracellular DNA and (ii) and/or extracellular RNA as separate fractions. Furthermore, there is a need for methods that can be automated. It is the object of the present disclosure to provide kits and methods that avoid drawbacks of the prior art. In particular, it is an object to prove a method and kit for enriching (i) extracellular DNA and (ii) EVs and/or extracellular RNA that comprises vesicular RNA SUMMARY OF THE DISCLOSURE The present disclosure is based on the finding that extracellular DNA from a biological sample comprising extracellular vesicles can be bound to a solid phase comprising anion exchange groups under conditions, wherein binding of EVs and/or cfRNA to the solid phase is reduced. As is demonstrated by the examples, the method according to the present disclosure allows to enrich extracellular DNA (also referred to as “cell-free DNA” or “cfDNA”) from biological samples comprising extracellular DNA and extracellular vesicles (EVs) (in particular cell-depleted body fluids such as plasma) by selectively binding extracellular DNA to the anion-exchange surface of a solid phase (e.g. magnetic beads) and separating the bound DNA from the remaining binding mixture. During the cfDNA binding step, binding of EVs to the solid phase can be reduced by choice of the binding conditions and the anion exchange groups of the solid phase, in particular by adjusting the acidic pH of the used binding buffer. As is demonstrated by the examples, different buffering agents and anion exchange groups can be used for preferential binding of cfDNA, while binding of EVs or cfRNA (also referred to as “cell-free RNA” or “extracellular RNA”) is reduced. In embodiments, at least 50% of the EVs and cfRNA remains in the binding mixture after separation of the solid phase with the bound cfDNA. The binding conditions according to the present disclosure thus allow to selectively capture extracellular DNA to the anion exchange surface without simultaneously capturing EVs (which contain most of the extracellular RNA) to the same extent to the anion exchange surface. This allows to provide an improved method for isolating extracellular DNA, because RNA contamination can be reduced. Moreover, these cfDNA selective binding conditions which reduce binding of EVs and cfRNA to the anion exchange surface provides the opportunity for the sequential isolation of different target analytes, e.g. cfDNA in the first binding step, followed by enrichment of EVs and/or total cfRNA in a second binding step. The cfDNA binding conditions used according to the invention advantageously do not promote the destruction of EVs. After separating the solid phase with the bound cfDNA, intact EVs and other analytes such as non-vesicular RNA (e.g. certain miRNAs) are comprised in the remaining binding mixture (e.g. supernatant). The remaining binding mixture can thus be used for isolation of other analytes, such as EVs and/or total cfRNA. This allows to provide cfDNA and vesicular RNA (or total cfRNA comprising vesicular RNA) in separate fractions, facilitating the separate analysis of the obtained fractions (e.g. eluates) using different types of assays. In this way, the present disclosure also provides methods for the sequential enrichment of extracellular DNA and other target analytes, such as the sequential enrichment of ccfDNA and EVs (or