PRE-TEMPLATED INSTANT PARTITIONING OF DNA-ENCODED LIBRARIES
This disclosure provides a powerful screening platform that combines pre-templated instant partitions with DNA-encoded library (DEL) technologies to identify target small molecule interactions and analyze their intracellular effects in single cell resolution using methods that require minimal sample preparation and affordable sequencing costs.
1 . A method for screening DNA-encoded libraries (DELs) with single cells, the method comprising:
binding cells with DELs comprising small molecules linked to DNA tags;
combining, in a tube containing a first fluid, the cells with template particles comprising capture oligos;
adding a second fluid immiscible with the first fluid to the tube;
shearing the fluids to create a plurality of partitions, near simultaneously, inside the tube, wherein a substantial number of the partitions contain a single one of the cells and a single one of the template particles; and
barcoding, with the capture oligos, mRNA and DNA tags released by the single cells inside the partitions for single cell analysis.
2 . The method of claim 1 , further comprising pre-screening candidate DELs in a binding assay and, based on the binding assay, selecting the DELs use for cell binding.
3 . The method of claim 2 , wherein the binding assay comprises:
combining a plurality of candidate DELs with targets;
enriching candidate DELs bound with targets; and
identifying a subset of the enriched candidate DELs for single cell analysis.
4 . The method of claim 3 , wherein the subset comprises a portion of the enriched candidate DELs having a higher target binding affinity than a second portion of the enriched candidate DELs.
5 . The method of claim 3 , wherein identifying the subset comprises sequencing DNA tags corresponding with the enriched candidate DELs to generate sequence reads.
6 . The method of claim 5 , further comprising quantifying the sequences reads of DELs to assess binding affinities, wherein a greater number of unique reads correlates to a higher binding affinity.
7 . The method of claim 5 , wherein the DNA tags comprise a barcode and a primer binding sequences.
8 . The method of claim 1 , wherein each one of the DNA tags comprises an oligonucleotide comprising at least one barcode sequence that is unique to the small molecule of the DEL.
9 . The method of claim 1 , wherein the small molecule comprises a drug candidate against a cell surface receptor.
10 . The method of claim 1 , wherein the oligos of the template particles comprise one or more of barcodes, primer binding sequences, or molecular binders for capturing mRNA or DNA tags released from cells.
11 . The method of claim 10 , wherein at least a portion of the oligos comprise molecular binders comprising poly-T capture sequences.
12 . The method of claim 1 , further comprising amplifying the barcoded mRNA and DNA tags released by the cells to generate amplicons.
13 . The method of claim 12 , wherein amplifying is performed with gene specific primers to thereby amplify mRNA associated with genes of interest.
14 . The method of claim 13 , wherein the genes of interests comprise genes involved in gene expression pathways that associated with drug candidates.
15 . The method of claim 12 , further comprising sequencing the amplicons to produce a plurality of sequence reads.
16 . The method of claim 15 , wherein the sequence reads comprise sequence information linking the small molecules of DELs with transcriptional output of corresponding single cells.
17 . The method of claim 1 , further comprising washing the cells after binding.
18 . The method of claim 1 , wherein the small molecules bind to surface receptors of the cells.
19 . The method of claim 1 , wherein binding involves integrating at least a portion of the DELs inside the cells.
20 . The method of claim 1 , wherein the DELs further comprise cell-penetrating ligands.
21 . The method of claim 1 , wherein the DELs target cell type specific proteins.