Granted Patent B2

US 12,203,139 · App. 18/160,918 · Granted Jan 21, 2025

Inventor: Timothy Rand (Chicago, IL)

Assignee: TEMPUS AI, INC.

C12Q1/6883C12Q1/6813

Abstract

Claims (44)

1. A method comprising:

a) providing a first antibody-oligo tag (AOT) comprising a first antibody and a first oligonucleotide, and a second AOT comprising a second antibody and a second oligonucleotide;

wherein the first antibody of the first AOT binds a first target in a biological sample, and the second antibody of the second AOT binds a second target in the biological sample;

wherein the first oligonucleotide of the first AOT comprises a first hybridization region, and the second oligonucleotide of the second AOT comprises a second hybridization region; and

wherein the first oligonucleotide of the first AOT comprises a restriction enzyme recognition site for BstXI and said site is located 5′ to the first hybridization region;

b) providing a splint oligonucleotide,

wherein the splint oligonucleotide comprises a first complementary splint region (CSR) that is complementary to the first hybridization region of the first AOT and a second CSR that is complementary to the second hybridization region of the second AOT, wherein the first and second hybridization regions hybridize to the first and second CSRs under hybridization conditions thereby forming a proximity detection nucleic acid (PDNA) that comprises at least one double-stranded portion and at least two single-stranded portions;

c) contacting, in a reaction vessel, the biological sample and the first and second AOTs under conditions that allow for binding of the first and second antibodies to their respective targets;

d) adding the splint oligonucleotide to the reaction vessel;

e) providing hybridization conditions to the reaction vessel wherein the hybridization regions of the first and second oligonucleotides of the first and second AOTs hybridize to the first and second CSRs of the splint oligonucleotide and form the PDNA if the first and second targets are in proximity in the biological sample;

f) contacting the PDNA with a DNA polymerase and deoxyribonucleotide triphosphates (dNTPs) to fill in the single-stranded portions of the PDNA, thereby forming a double-stranded PDNA;

g) cutting the double-stranded PDNA with BstXI to form a cut PDNA comprising a single-stranded 3′-overhang; and

h) detecting the cut PDNA using single-cell sequencing.

2. A method comprising:

a) providing a first antibody-oligo tag (AOT) comprising a first antibody and a first oligonucleotide, and a second AOT comprising a second antibody and a second oligonucleotide;

wherein the first antibody of the first AOT binds a first target in a biological sample, and the second antibody of the second AOT binds a second target in the biological sample;

wherein the first oligonucleotide of the first AOT comprises a first and a second hybridization region, and the second oligonucleotide of the second AOT comprises a third and a fourth hybridization region; and

wherein the first oligonucleotide of the first AOT comprises a restriction enzyme recognition site for BstXI and said site is located 5′ to the first hybridization region;

b) providing a primer oligonucleotide comprising a primer region and a region complementary to the first hybridization region of the first AOT;

c) providing a capture oligonucleotide comprising a capture region and a region complementary to the third hybridization region of the second AOT;

d) providing a splint oligonucleotide comprising

a first complementary splint region (CSR), wherein the first CSR is complementary to the second hybridization region of the first AOT, and

a second CSR, wherein the second CSR is complementary to the fourth hybridization region of the second AOT;

e) contacting, in a reaction vessel, the biological sample and the first and second AOTs under conditions that allow for binding the first and second antibodies to their respective targets;

f) adding the primer oligonucleotide, the capture oligonucleotide, and the splint oligonucleotides to the reaction vessel;

g) providing hybridization conditions in the reaction vessel wherein the first and second hybridization regions of the first AOT and the third and fourth hybridization regions of the second AOT hybridize to the primer oligonucleotide, the capture oligonucleotide, and the splint oligonucleotide to form a PDNA if the first and second targets are in proximity in the biological sample;

h) contacting the PDNA with a DNA polymerase and deoxyribonucleotide triphosphates (dNTPs) to fill in the single-stranded portions of the PDNA, thereby forming a double-stranded PDNA;

i) cutting the double-stranded PDNA with BstXI to form a cut PDNA comprising a single-stranded 3′-overhang; and

j) detecting the cut PDNA using single-cell sequencing.

3. The method of claim 1 , wherein the first and second antibodies bind to: (i) different targets on the same molecule, or (ii) different targets on different molecules.

4. The method of claim 1 , wherein at least one of the first or second targets comprises a protein, a sugar, or a phosphate.

5. The method of claim 1 , wherein at least one of the first or second targets is a cancer biomarker and is expressed on the surface of a cancer cell.

6. The method of claim 1 , wherein at least one of the first or second targets is selected from the group consisting of CD274, CD16, CD56, CD4, CD8a, CD19, CD20, CD21, CD19, CD25, CD279, CD278, CD137, CD127, CD273, CD14, CD117, CD152, CD223, CD134, CD141, CD34, CD45, and CD3.

7. The method of claim 1 , wherein the first and second AOT oligonucleotides comprise one or more barcode sequences.

8. The method of claim 1 further comprising providing at least one mask oligonucleotide in step (a), wherein the sequence of the mask oligonucleotide is complementary to at least one of the hybridization regions on the first or second oligonucleotide of the first or second AOT.

9. The method of claim 1 , wherein step (f) further comprises providing single-cell sequencing data to a system, the system comprising:

at least one processor that is programmed to:

receive single-cell sequencing data comprising a barcodes file and a features file;

generate FASTQ files from the single-cell sequencing data;

define a plurality of search strings, wherein each search string in the plurality of search strings comprises at least two feature site sequences and at least one spacer of a defined length, wherein the at least two feature site sequences are associated with the first and second targets;

determine a location of each search string in the plurality of search strings in the FASTQ files and unique identifiers of the determined location;

cause the determined location and the unique identifiers of each search string in the plurality of search strings to be presented.

10. The method of claim 9 , wherein a search string in the plurality of search strings is associated with a sequence of the PDNA.

11. The method of claim 9 , wherein a search string in the plurality of search strings is associated with a segment of the sequence of the PDNA.

Assignments (4)

Continuity (3)

References Cited (79)