Methods and compositions for delivery of molecules and complexes to reaction sites

Abstract

The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in an active loading of molecules and complexes into reaction sites with improved efficiency over loading by passive diffusion methods alone.

1 . A method of delivering a molecule to a site, the method comprising:
 (a) providing a tethering nucleic acid at the site, wherein at least a portion of the tethering nucleic acid is located outside of the site, wherein the tethering nucleic acid comprises (i) a first strand comprising a first capture moiety at a position that is located outside of the site, and (ii) a second strand;   (b) exposing the tethering nucleic acid to a molecule comprising a first binding moiety, wherein the first binding moiety and the first capture moiety interact to attach the molecule to the tethering nucleic acid; and   (c) degrading the second strand of the tethering nucleic acid, wherein the first strand contains self-complementary regions that hybridize together to collapse the first strand into a hairpin, wherein the collapse of the first strand further serves to carry the attached molecule into the interior of the site,   thereby delivering the molecule to the site.   
     
     
         2 . The method of  claim 1 , wherein the first capture moiety comprises biotin and the first binding moiety comprises streptavidin. 
     
     
         3 . The method of  claim 1 , wherein the second strand comprises RNA, and wherein in step (c) degrading the second strand comprises applying RNAse to the tethering nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein the second strand comprises at least one deoxyuridine (dU), and wherein in step (c) degrading the second strand comprises applying uracil-DNA glycosylase and endonuclease VIII to the tethering nucleic acid. 
     
     
         5 . The method of  claim 1 , wherein the first strand is resistant to exonuclease and the second strand is sensitive to exonuclease, and wherein in step (c) degrading the second strand comprises applying an exonuclease to the tethering nucleic acid. 
     
     
         6 . The method of  claim 1 , wherein the molecule is a member selected from the group consisting of: a template nucleic acid, a polypeptide, an antibody, and a small molecule. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the molecule is part of a complex. 
     
     
         9 . The method of  claim 8 , wherein the molecule comprises a template nucleic acid complexed with a polymerase enzyme. 
     
     
         10 . The method of  claim 9 , wherein the complex further comprises a primer hybridized to the template nucleic acid. 
     
     
         11 . The method of  claim 9 , wherein the first binding moiety is associated with the polymerase enzyme. 
     
     
         12 . The method of  claim 1 , wherein the first strand of the tethering nucleic acid comprises the first capture moiety at one end and a second capture moiety at the other end. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 12 , wherein the first and second capture moieties are different. 
     
     
         15 . The method of  claim 12 , wherein the site comprises a second binding moiety, and wherein the tethering nucleic acid is immobilized to the site by an interaction between the second capture moiety and the second binding moiety. 
     
     
         16 . The method of  claim 1 , wherein the tethering nucleic acid is at least 1000 nucleotides in length. 
     
     
         17 . The method of  claim 1 , wherein the site comprises a nano scale well. 
     
     
         18 . The method of  claim 1 , wherein the site is at the base of a nanoscale well. 
     
     
         19 . The method of  claim 1 , wherein the site comprises a nanopore. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the method further comprises step (d) immobilizing the molecule to the site. 
     
     
         22 . The method of  claim 21 , wherein the immobilizing comprises an interaction between a first reaction moiety on the site and a second reaction moiety on the molecule. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the first capture moiety comprises a scaffold, wherein the scaffold comprises (i) a core comprising conjugation adaptors and (ii) multiple arms comprising biotin moieties. 
     
     
         25 - 105 . (canceled)