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 loading of polymerase enzyme complexes into a predetermined number of reaction sites, including nanoscale wells.

1 . A method for loading polymerase enzyme complexes into a predetermined number of nanoscale wells, the method comprising:
 (a) providing a surface comprising an array of nanoscale wells;   (b) contacting a loading solution to the surface, wherein the loading solution comprises:
 (i) one or more nucleotides and/or nucleotide analogs; and 
 (ii) polymerase enzyme complexes comprising a template nucleic acid and a polymerase enzyme, 
   wherein interactions between the nucleotides and/or nucleotide analogs and the polymerase enzyme complexes result in generation of signal pulses;   (c) while the loading solution is in contact with the surface, monitoring the array of nanoscale wells to detect signal pulses from within the wells and thereby identifying nanoscale wells that have been loaded with a polymerase enzyme complex at one or more time points after contacting the loading solution to the surface, to obtain initial loading data;   (d) from the initial loading data, calculating a predicted end time point at which the predetermined number of nanoscale wells will have been loaded with a polymerase enzyme complex; and   (e) maintaining the loading solution in contact with the surface until the predicted end time point is reached, thereby loading the predetermined number of nanoscale wells with a polymerase enzyme complex.   
     
     
         2 . The method of  claim 1 , wherein the loading solution comprises one or more labeled nucleotide analogs, wherein the signal pulses are a result of non-incorporation events involving the labeled nucleotide analogs. 
     
     
         3 . The method of  claim 2 , wherein the loading solution comprises one or more additives that maintain the polymerase enzymes in an inactive state. 
     
     
         4 . The method of  claim 3 , wherein the one or more additives comprise a divalent cation selected from the group consisting of strontium, cobalt, tin, calcium, nickel, europium, barium, iron, and zinc. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 2 , wherein the labeled nucleotide analog is a nonhydrolyzable labeled nucleotide analog. 
     
     
         7 . The method of  claim 1 , wherein the array of nanoscale wells is part of a substrate that allows signal pulses to be detected only when a polymerase enzyme complex is within a nanoscale well. 
     
     
         8 . The method of  claim 1 , wherein the template nucleic acids in the polymerase enzyme complexes are hybridized to a primer. 
     
     
         9 . The method of  claim 1 , wherein the template nucleic acids comprise a hairpin loop at one or both ends. 
     
     
         10 . The method of  claim 1 , wherein the predetermined number of nanoscale wells occupied by a polymerase enzyme complex is about 60-80% of the nanoscale wells in the array. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 26 , wherein after the wash step, the array is prepared for analyzing the polymerase enzyme complexes within the nanoscale wells. 
     
     
         13 . The method of 12, wherein the analyzing comprises determining a nucleotide sequence of the template nucleic acid. 
     
     
         14 . The method of  claim 13 , wherein determining the nucleotide sequence comprises
 (i) providing one or more nucleotides or nucleotide analogs;   (ii) performing a polymerization reaction in which the polymerase enzyme replicates at least a portion of the template nucleic acid in a template-dependent manner, whereby one or more of the nucleotides or nucleotide analogs are incorporated into the resulting nucleic acid; and   (iii) identifying a time sequence of incorporation of the one or more nucleotide or nucleotide analogs into the resulting nucleic acid.   
     
     
         15 . The method of  claim 1 , comprising immobilizing the polymerase enzyme complexes within the nanoscale wells. 
     
     
         16 . The method of  claim 15 , wherein the polymerase enzyme complexes comprise a reactive element, and the immobilizing occurs through an interaction of the reactive element and a binding site in the nanoscale well. 
     
     
         17 . The method of  claim 16 , wherein the reactive element comprises streptavidin and the binding site comprises biotin. 
     
     
         18 . The method of  claim 1 , wherein the loading solution further comprises at least one agent to mitigate photodamage. 
     
     
         19 .- 20 . (canceled) 
     
     
         21 . The method of  claim 18 , wherein the at least one agent to mitigate photodamage is selected from the group consisting of a triplet-state quencher, a reducing agent, a singlet oxygen quencher, and an oxygen depleting enzyme. 
     
     
         22 .- 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein monitoring the array of nanoscale wells to detect signal pulses from within the wells and thereby identifying nanoscale wells that have been loaded with a polymerase enzyme complex comprises monitoring the array of nanoscale wells to detect signal pulses from within the wells and thereby identifying nanoscale wells that have been loaded with a polymerase enzyme complex at 1-10 time points after contacting the loading solution to the surface, to obtain initial loading data. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1 , comprising washing the surface to remove the loading solution at the predicted end time point. 
     
     
         27 - 158 . (canceled) 
     
     
         159 . The method of  claim 1 , wherein monitoring the array of nanoscale wells to detect signal pulses from within the wells and thereby identifying nanoscale wells that have been loaded with a polymerase enzyme complex comprises monitoring the array of nanoscale wells to detect signal pulses from within the wells and thereby identifying nanoscale wells that have been loaded with a polymerase enzyme complex at 2-10 time points after contacting the loading solution to the surface, to obtain initial loading data.