Methods and systems for microfluidic screening

Abstract

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors.

1 . A method for amplifying a primer to maximize cellular nucleic acid capture, the method comprising:
 (a) providing an encapsulation encapsulating a scaffold, one or more cells, an amplification mix, and a nicking enzyme, wherein a nucleic acid encoding is bound to the scaffold;   (b) lysing the one or more cells to release one or more cellular nucleic acids;   (c) nicking the nucleic acid encoding with the nicking enzyme, thereby creating a first encoded nucleic acid primer;   (d) amplifying the first encoded nucleic acid primer via the nicking site and amplification mix; and   (e) labeling the one or more cellular nucleic acids with the first encoded nucleic acid primer.   
     
     
         2 . The method of  claim 1 , wherein the nicking enzyme targets a specific site in the nucleic acid encoding, wherein the specific site comprises a specific nucleotide sequence. 
     
     
         3 . The method of  claim 1 , wherein the amplifying of d) comprises i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) nicking the copy of the nucleic acid encoding to create a second encoded nucleic acid primer. 
     
     
         4 . The method of  claim 1 , wherein the amplifying of d) comprises simultaneously i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) displacing the copy of the nucleic acid encoding to create a second encoded nucleic acid primer. 
     
     
         5 . The method of  claim 4 , wherein the amplification mix comprises an amplification enzyme, such that the amplification enzyme enables the copy of the nucleic acid encoding to be simultaneously created and displaced. 
     
     
         6 . The method of  claim 5 , wherein the amplification enzyme comprises a polymerase. 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid encoding comprises a capture site that prescribes a target cellular nucleic acid to label the one or more cellular nucleic acids. 
     
     
         8 . The method of  claim 7 , wherein the target cellular nucleic acid is a target mRNA. 
     
     
         9 . The method of  claim 1 , wherein the scaffold comprises a bead, and the first encoded nucleic acid primer comprises a unique bead barcode and an encoding corresponding to an encoded effector. 
     
     
         10 . The method of  claim 1 , wherein the scaffold comprises an encoded effector bound thereto by a cleavable linker. 
     
     
         11 . The method of  claim 1 , wherein the labeling of e) comprises barcoding the one or more cellular nucleic acids. 
     
     
         12 . The method of  claim 1 , further comprising performing an effector screen, wherein the one or more cells are being screened against an encoded effector. 
     
     
         13 . A system for amplifying a primer to maximize cellular nucleic acid capture, the system comprising:
 (a) a scaffold, wherein a nucleic acid encoding is bound to the scaffold;   (b) one or more cells each comprising one or more cellular nucleic acids;   (c) an amplification mix;   (d) a nicking enzyme;   (e) a microfluidic device configured to:
 (i) receive the scaffold, the one or more cells, the amplification mix, and the nicking enzyme, 
 (ii) encapsulate the scaffold, the one or more cells, the amplification mix, and the nicking enzyme in an encapsulation, 
 (iii) lyse the one or more cells, such that the one or more cellular nucleic acids are released, 
 (iv) nick the nucleic acid encoding with the nicking enzyme, thereby creating a first encoded nucleic acid primer, wherein the one or more cellular nucleic acids are labeled with the first encoded nucleic acid primer, and 
 (v) amplify the first encoded nucleic acid primer. 
   
     
     
         14 . The system of  claim 13 , wherein the nicking enzyme is configured to target a specific site in the nucleic acid encoding, wherein the specific site comprises a specific nucleotide sequence. 
     
     
         15 . The system of  claim 13 , wherein the encoded nucleic acid primer is amplified by i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) nicking the copy of the nucleic acid encoding to create a second encoded nucleic acid primer. 
     
     
         16 . The system of  claim 13 , wherein the encoded nucleic acid primer is amplified by simultaneously i) creating a copy of the nucleic acid encoding that extends from the nicking site; and ii) displacing the copy of the nucleic acid encoding to create a second encoded nucleic acid primer. 
     
     
         17 . The system of  claim 16 , wherein the amplification mix comprises an amplification enzyme, such that the amplification enzyme enables the copy of the nucleic acid encoding to be simultaneously created and displaced. 
     
     
         18 . The system of  claim 13 , wherein each nucleic acid encoding comprises a capture site that prescribes a target cellular nucleic acid to label the one or more cellular nucleic acids. 
     
     
         19 . The system of  claim 13 , wherein the scaffold comprises a bead, and the first encoded nucleic acid primer comprises a unique bead barcode and an encoding corresponding to an encoded effector. 
     
     
         20 . The system of  claim 13 , wherein the one or more cellular nucleic acids are labelled with the first encoded nucleic acid primer through barcoding of the one or more cellular nucleic acids, wherein the encapsulation further comprises barcoding reagents.