Methods for obtaining information from single cells within populations using DNA origami nanostructures without the need for single cell sorting
Methods for construction of DNA origami nanostructures, as well as for binding, isolation, linking, and deep sequencing information, such as both of TCR alpha and beta CDR3 mRNA, from individual cells within a mixed population of cells without the need for single cell sorting (FIG. 1 ).
1. A method for obtaining genetic information from an individual cell, comprising the steps of:
(a) transfecting a cell with a DNA origami nanostructure comprising a first polynucleotide sequence complementary to a first target RNA sequence and a second polynucleotide sequence complementary to a second target RNA sequence;
(b) isolating said DNA origami nanostructure from said transfected cell, wherein the DNA origami nanostructure is bound to a first and second complementary RNA from said cell;
(c) reverse transcribing said first and second complementary RNA into complementary DNA (cDNA); and
(d) sequencing the cDNA.
2. The method of claim 1 , wherein said cell is a primary T cell;
wherein said first target sequence comprises a TCR alpha mRNA constant region and said second target sequence comprises a TCR beta mRNA constant region; wherein said DNA origami nanostructure comprises a ssDNA M13 phage genome refolded with complementary ssDNA staple sequences into a predetermined shape with least two staples extended with DNA sequences complementary to, respectively, a TCR alpha constant region mRNA and a TCR beta constant region mRNA; and wherein the first complementary RNA is a TCR alpha mRNA and the second complementary RNA is a TCR beta mRNA.
3. The method of claim 1 , wherein the DNA origami nanostructure further comprises a single-stranded DNA (ssDNA) having an M13 phage sequence.
4. The method of claim 1 , wherein transfecting comprises electroporation.
5. The method of claim 1 , wherein isolating comprises lysis of the cell.
6. The method of claim 1 , wherein the DNA origami nanostructure further comprises a biotin tag.
7. The method of claim 6 , wherein the isolating step comprises a streptavidin purification.
8. The method of claim 7 , wherein the streptavidin purification comprises contacting the DNA origami nanostructure bound to the first and second complementary RNA with a streptavidin chromatography column.
9. The method of claim 1 , wherein reverse transcribing comprises contacting the DNA origami nanostructure with a reverse transcriptase lacking exonuclease activity.
10. The method of claim 9 , wherein reverse transcribing comprises an RNase inhibitor to reduce displacement activity of the reverse transcriptase.
11. The method of claim 1 , wherein after the reverse transcribing step and prior to the sequencing step the method further comprises ligating the cDNA produced in step (c) to form a single cDNA.
12. The method of claim 11 , wherein the ligating comprises contacting the isolated DNA origami nanostructure with a T4 DNA ligase.
13. The method of claim 11 , wherein after the ligating step and prior to the sequencing step the method further comprises a nucleic acid amplification step.
14. The method of claim 13 , wherein the sequencing comprises high-throughput sequencing.
15. The method of claim 1 , wherein the DNA origami nanostructure further comprises a fluorescent dye.
16. The method of claim 1 , wherein after the reverse transcribing step and prior to the sequencing step the method further comprises a nucleic acid amplification step.
17. The method of claim 16 , wherein the nucleic acid amplification comprises a multiplex polymerase chain reaction (PCR) amplification using a Cβ primer and a multiplex of Vα primers.
18. The method of claim 17 , wherein the sequencing comprises high-throughput sequencing.
19. The method of claim 2 , wherein the sequencing comprises CDR3 paired end sequencing.