Granted Patent B2

US 12,631,624 · App. 17/287,341 · Granted May 19, 2026

Inventors: David Routenberg (Gaithersburg, MD); Alexander K. Tucker-Schwartz (Bethesda, MD); Sigal Shachar (North Bethesda, MD)

Assignee: MESO SCALE TECHNOLOGIES, LLC.

G01N33/54306C12N15/1065C12Q1/6834

Abstract

Claims (25)

1 . A method of determining surface markers of a surface marker displaying agent, comprising:

a. contacting a sample comprising a surface marker displaying agent with:

(i) a first binding reagent comprising a first oligonucleotide comprising a first unique barcode sequence and a first hybridization sequence;

(ii) a second binding reagent comprising a second oligonucleotide comprising a second hybridization sequence, a second unique barcode sequence, and a 5′ splint complement sequence;

(iii) a third binding reagent comprising a third oligonucleotide comprising a 3′ splint complement sequence, a third unique barcode sequence, and a third hybridization sequence;

(iv) a splint oligonucleotide; and

(v) a capture reagent, wherein the capture reagent is linked to an anchoring reagent, wherein the anchoring reagent is releasably bound to a surface and comprises:

a first cleavage site;

a second cleavage site; and

a fourth oligonucleotide comprising a fourth hybridization sequence and a fourth unique barcode sequence,

wherein the first hybridization sequence and the second hybridization sequence are complementary, the third hybridization sequence and the fourth hybridization sequence are complementary, and the 5′ splint complement sequence and the 3′ splint complement sequence are respectively complementary to 5′ and 3′ ends of the splint oligonucleotide,

wherein if the surface marker displaying agent binds to the capture reagent linked to the anchoring reagent, and to the first, second, and third binding reagents, then an output oligonucleotide is generated that comprises the first, second, third, and fourth unique barcode sequences, and

wherein the first cleavage site is configured to be cleaved under conditions at which the second cleavage site is not cleaved;

b. cleaving at the first cleavage site and eluting unwanted components from the sample, wherein the surface marker displaying agent is retained on the surface after cleaving at the first cleavage site; and

c. sequencing the output oligonucleotide to identify the first, second, third, and fourth barcode sequences therein, thereby determining at least four unique surface markers of the surface marker displaying agent.

2 . The method of claim 1 , wherein the first oligonucleotide further comprises a first primer site, and the fourth oligonucleotide further comprises a second primer site.

3 . The method of claim 1 , wherein at least one cleavage site is a restriction site.

4 . The method of claim 1 , wherein the capture reagent is linked to the anchoring reagent with polyethylene glycol (PEG) or with a polynucleotide sequence.

5 . The method of claim 4 , wherein the capture reagent is linked to the anchoring reagent with a polynucleotide sequence, wherein the polynucleotide sequence comprises poly(A).

6 . The method of claim 1 , wherein the surface marker displaying agent is a cell, a virus or viral particle, an organelle, a vesicle, or combination thereof.

7 . The method of claim 1 , further comprising cleaving at the second cleavage site to release the surface marker displaying agent from the surface.

8 . The method of claim 1 , further comprising adding adaptor sequences to the output oligonucleotide for PCR amplification and sequencing.

9 . The method of claim 1 , wherein the anchoring reagent further comprises a surface attachment moiety.

10 . The method of claim 9 , wherein the surface attachment moiety comprises biotin.

11 . The method of claim 1 , wherein the surface comprises streptavidin.

Assignments (1)

Priority Claims (1)

Continuity (2)

References Cited (85)