Method for attaching one or more polynucleotide binding proteins to a target polynucleotide

Abstract

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also related to new methods of characterising target polynucleotides.

1 . A method for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising:
 (a) providing the one or more polynucleotide binding proteins bound to one or more loading moieties; and   (b) attaching the one or more loading moieties to the target polynucleotide.   
     
     
         2 . A method according to  claim 1 , wherein the method comprises before step (a) binding the polynucleotide binding proteins to the one or more loading moieties. 
     
     
         3 . A method according to  claim 1 , wherein the one or more polynucleotide binding proteins are derived from one or more polynucleotide handling enzymes. 
     
     
         4 . A method according to  claim 3 , wherein the one or more polynucleotide handling enzymes are one or more polymerases, exonucleases, helicases, topoisomerases or a combination thereof. 
     
     
         5 . A method according to  claim 4 , wherein the one or more helicases are a) Hel308 helicases, RecD helicases, XPD helicases or Dda helicases (b) helicases derived from any of the helicases in (a); or (c) a combination of any of the helicases in (a) and/or (b). 
     
     
         6 . A method according to  claim 5 , wherein the one or more helicases are modified to reduce the size of an opening in the polynucleotide binding domain through which in at least one conformational state the polynucleotide can unbind from the helicase. 
     
     
         7 .- 9 . (canceled) 
     
     
         10 . A method according to  claim 1 , wherein the one or more polynucleotide binding proteins remain bound to the one or more loading moieties at the end of the step (b). 
     
     
         11 .- 13 . (canceled) 
     
     
         14 . A method according to  claim 5 , wherein the one or more polynucleotide binding proteins are derived from helicases are stalled at one or more spacers on the one or more loading polynucleotides. 
     
     
         15 .- 24 . (canceled) 
     
     
         25 . A method of characterising a target polynucleotide, comprising:
 (a) carrying out a method according to  claim 1 ;   (b) contacting the target polynucleotide having the one or more attached polynucleotide binding proteins as provided in step (a) with a transmembrane pore such that the one or more polynucleotide binding proteins control the movement of the polynucleotide with respect to the pore; and   (c) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.   
     
     
         26 . A method of preparing a target polynucleotide for characterisation, comprising:
 (a) carrying out a method according to  claim 1  wherein the one or more polynucleotide binding proteins comprise one or more polymerases; and   (b) allowing the one or more polymerases attached to the target polynucleotide provided in step (a) to form one or more polynucleotides using the target polynucleotide as a template and thereby preparing the target polynucleotide for characterisation.   
     
     
         27 . A method of characterising a target polynucleotide, comprising:
 (a) carrying out a method according to  claim 26 ;   (b) contacting the target polynucleotide and/or the one or more polynucleotides produced in step (a) with a transmembrane pore such that the target polynucleotide and/or the one or more polynucleotides produced in step (a) move with respect to the pore; and   (c) taking one or more measurements as the target polynucleotide and/or the one or more polynucleotides produced in step (a) move with respect to the pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and/or the one or more polynucleotides produced in step (a) and thereby characterising the target polynucleotide.   
     
     
         28 . A method according to  claim 1 , wherein the pore is a transmembrane protein pore or a solid state pore. 
     
     
         29 . A method according to  claim 28 , wherein the transmembrane protein pore is derived from a hemolysin, leukocidin,  Mycobacterium smegmatis  porin A (MspA), MspB, MspC, MspD, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A,  Neisseria  autotransporter lipoprotein (NalP) and WZA. 
     
     
         30 .- 32 . (canceled) 
     
     
         33 . A method according to  claim 25 , wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement. 
     
     
         34 . A method according to  claim 33 , wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (PET) measurement. 
     
     
         35 . A target polynucleotide modified using a method according to  claim 1 . 
     
     
         36 . (canceled) 
     
     
         37 . A kit for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising (a) the one or more polynucleotide binding proteins bound to one or more loading moieties and (b) a ligase. 
     
     
         38 . (canceled)