Enzyme stalling method

Abstract

The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.

1 . (canceled) 
     
     
         2 . A method of controlling the movement of a target polynucleotide through a transmembrane pore, comprising:
 (a) providing the target polynucleotide with one or more spacers;   (b) contacting the target polynucleotide with one or more helicases such that the one or more helicases stall at the one or more spacers;   (c) contacting the target polynucleotide and the one or more stalled helicases with the pore; and   (d) applying a potential across the pore such that the one or more helicases move past the one or more spacers and control the movement of the target polynucleotide through the pore.   
     
     
         3 . A method of characterising a target polynucleotide, comprising:
 (a) carrying out the method of  claim 2 ; and   (b) 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.   
     
     
         4 . A method according to  claim 2 , wherein providing the target polynucleotide with one or more spacers comprises modifying the target polynucleotide so that it comprises one or more spacers. 
     
     
         5 . A method according to  claim 3 , wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether or not the target polynucleotide is modified. 
     
     
         6 . A method according to  claim 5 , wherein the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. 
     
     
         7 . A method according to  claim 3 , wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement. 
     
     
         8 . A method according to  claim 7 , wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement. 
     
     
         9 . A method according to  claim 2 , wherein the one or more helicases control the movement of the target polynucleotide through the pore with the field resulting from the applied potential. 
     
     
         10 . A method according to  claim 2 , wherein the one or more spacers have a different structure from the polynucleotide. 
     
     
         11 . A method according to  claim 2 , wherein the polynucleotide is desoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and the one or more spacers comprise peptide nucleic acid (PNA), glycerol nucleic acid (ONA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains. 
     
     
         12 . A method according to  claim 2 , wherein the one or more spacers comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one or more dideoxy-cytidines (ddCs), one m more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2′-O-Methyl RNA bases, one or more Iso-deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines (Iso-dGs), one or more C3 groups, one or more photo-cleavable (PC) groups, one or more hexandiol groups, one or more spacer  9  (iSp9) groups, one or more spacer  18  (iSp18) groups, a polymer or one or more thiol connections. 
     
     
         13 - 33 . (canceled) 
     
     
         34 . A method of controlling the loading of one or more helicases on a target polynucleotide, comprising:
 (a) providing the target polynucleotide with one or more spacers; and   (b) contacting the target polynucleotide provided in (a) with the one or more helicases such that the one or more helicases bind to the target polynucleotide and stall at each spacer.   
     
     
         35 - 48 . (canceled) 
     
     
         49 . A kit for controlling the movement of a target polynucleotide, wherein the kit comprises (a) one or more spacers, (b) one or more helicases and (c) a transmembrane pore. 
     
     
         50 . A kit according to  claim 49 , wherein the one or more spacers comprise one or more chemical groups which cause the one or more helicases to stall. 
     
     
         51 . A kit according to  claim 49 , wherein the kit further comprises an adaptor comprising:
 (a) (L-S-D)n or (D-S-L)n in the 5′ to 3′ direction, wherein L is a single stranded polynucleotide or a non-hybridised polynucleotide, S is a spacer and D is a double stranded polynucleotide and wherein n is a whole number, and   (b) one or more helicases stalled on each adaptor.   
     
     
         52 . The kit according to  claim 50 , wherein the one or more chemical groups are one or more fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, a dinitrophenol (DNP), digoxigenin and/or anti-digoxigenin or a dibenzylcyclooctyne group. 
     
     
         53 . The kit according to  claim 49 , wherein the one or more spacers are capable of stalling the one or more helicases in the presence of free nucleotides and/or the presence of a helicase cofactor. 
     
     
         54 . The kit according to  claim 49 , wherein the one or more spacers are capable of stalling the one or more helicases in a salt concentration of about 100 mM or lower. 
     
     
         55 . The kit according to  claim 51 , wherein L is capable of binding only one helicase (L) or capable of binding only two helicases (L 2 ). 
     
     
         56 . The kit according to  claim 51 , wherein n is 1 and one or two helicases are stalled on the adaptor.