Terminal functionalization of target molecules for sequencing

Patent metadata

Field	Value
Publication number	US-2021331170-A1
Application number	US-202117236889-A
Country	US
Kind code	A1
Filing date	Apr 21, 2021
Priority date	Apr 22, 2020
Publication date	Oct 28, 2021
Grant date	—

Abstract

Methods and devices for preparing target molecules (e.g., target nucleic acids or target proteins) from a biological sample are provided herein. In some embodiments, methods and devices involve sample lysis, sample fragmentation, enrichment of target molecule(s), and/or functionalization of target molecule(s).

First claim

What is claimed is:

1 . A device for preparing a biological sample for sequencing, wherein the device comprises an automated module configured to receive (iv) a functionalization cartridge comprising one or more microfluidic channels and configured to functionalize a terminal moiety of at least one of the one or more target molecules to form a functionalized sample; and one or more of the cartridges selected from (i) a lysis cartridge, (ii) an enrichment cartridge, and (iii) a fragmentation cartridge;
wherein (i), (ii), and (iii) are defined as follows:
(i) a lysis cartridge comprises one or more microfluidic channels and is configured to intake a biological sample comprising one or more target molecules and produce a lysed sample;
(ii) an enrichment cartridge comprises one or more microfluidic channels and is configured to enrich at least one of the one or more target molecules to produce an enriched sample; and
(iii) a fragmentation cartridge comprises one or more microfluidic channels and is configured to digest or fragment at least one of the one or more target molecules to produce a fragmented sample.

2 . The device of claim 1 , wherein the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, plant sample, blood sample, saliva sample, sputum sample, fecal sample, urine sample, buccal swab sample, amniotic sample, seminal sample, synovial sample, spinal sample, or pleural fluid sample.

3 . The device of claim 1 , wherein the one or more target molecules are nucleic acids or proteins.

4 . The device of claim 1 , wherein the one or more microfluidic channels are configured to contain and/or transport fluid(s) and/or reagent(s).

5 . The device of claim 1 , wherein the functionalization cartridge comprises a first chamber comprising reagents that covalently modify a moiety M 0 of the one or more target molecules, or of one or more fragments thereof, to a modified moiety M 1 .

6 . The device of claim 5 , wherein the reagents are non-enzymatic.

7 . The device of claim 5 , wherein the covalent modification is regiospecific.

8 . The device of claim 1 , wherein the portion of the one or more target molecules, or of the one or more fragments thereof, is a C-terminal carboxylate group or a C-terminal amino group.

9 . The device of claim 5 , wherein the reagents comprise buffers, salts, organic compounds, acids, and/or bases.

10 . The device of claim 5 , wherein the portion of the one or more target molecules, or of the one or more fragments thereof, is a C-terminal amino group, and the covalent modification is diazo transfer.

11 . The device of claim 10 , wherein moiety M 0 is —NH 2 and moiety M 1 is —N 3 .

12 . The device of claim 11 , wherein the reagents comprise imidazole-1-sulfonyl azide and a copper salt (e.g., copper sulfate), and a buffer having a pH of about 10-11.

13 . The device of claim 1 , wherein the first chamber is connected via one or more microfluidic channels, and/or optionally a purification chamber, to a second chamber.

14 . The device of claim 13 , wherein the second chamber comprises reagents that covalently modify moiety M 1 to produce a functionalized peptide.

15 . The device of claim 5 , wherein the covalent modification is an electrocyclic click reaction.

16 . The device of claim 15 , wherein the reagents comprise a DBCO-labeled DNA-streptavidin conjugate and a buffer, optionally wherein the DBCO-labeled DNA-streptavidin conjugate is immobilized to the surface of the second chamber.

17 . The device of claim 14 , wherein the functionalized peptide is functionalized with a DBCO-labeled DNA-streptavidin conjugate.

18 . The device of claim 13 , comprising a purification chamber positioned between the first chamber and the second chamber, comprising a resin that promotes purification or enrichment of the modified target molecules, or fragments thereof.

19 . The device of claim 18 , wherein the resin is Sephadex resin, optionally G-10 Sephadex resin.

20 . A device for preparing one or more target molecules, configured to perform step (iv) functionalize a terminal moiety of the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iii),
wherein (i), (ii), and (iii) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules;
(ii) enrich at least one of the one or more target molecules and/or at least one non-target molecule; and
(iii) fragment the one or more target molecules.

21 . A method for preparing one or more target molecules, comprising step (iv) functionalize a terminal moiety of the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iii),
wherein (i), (ii), and (iii) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules;
(ii) enrich at least one of the one or more target molecules and/or at least non-target molecule; and
(iii) fragment the one or more target molecules prior to functionalization;

wherein step (iv) is performed in an automated sample preparation device.

22 . A cartridge for preparing one or more target molecules, configured to perform step (iv) functionalize a terminal moiety of the one or more target molecules; and one or more of the following steps selected from (i), (ii), and (iii),
wherein (i), (ii), and (iii) are defined as follows:
(i) lyse a biological sample comprising one or more target molecules;
(ii) enrich at least one of the one or more target molecules and/or at least one non-target molecule; and
(iii) fragment the one or more target molecules.

Assignees

• Quantum Si Inc

Inventors

• Rothberg Jonathan M
• Leamon John H
• Schultz Jonathan C
• Millham Michele
• Lv Caixia
• Huang Haidong
• Nani Roger
• Ad Omer
• Reed Brian
• Dyer Matthew
• Boer Robert E

Classifications

• B01L2400/0481

  squeezing of channels or chambers · CPC title

• B01L2200/10

  Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept · CPC title

• B01L2200/16

  Reagents, handling or storing thereof · CPC title

• G01N33/6803 Primary

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• B01L3/502761 Primary

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title