Method for controlling the movement of a polynucleotide through a transmembrane pore

Abstract

The invention relates to new methods of controlling the movement of polynucleotides through transmembrane pores. The invention also relates to new methods of characterising target polynucleotides using helicases.

1 . A method for controlling the movement of a polynucleotide through a transmembrane pore, comprising:
 (a) providing the polynucleotide with one or more helicases attached to the polynucleotide and one or more molecular brakes attached to the polynucleotide;   (b) contacting the polynucleotide provided in step (a) with the pore; and   (c) applying a potential across the pore such that the one or more helicases and the one or more molecular brakes are brought together and both control the movement of the polynucleotide through the pore.   
     
     
         2 . A method according to  claim 1 , wherein the one or more molecular brakes comprise (a) one or more compounds which bind to the polynucleotide and/or (b) one or more proteins which bind to the polynucleotide. 
     
     
         3 . A method according to  claim 2 , wherein the one or more compounds are one or more macrocycles. 
     
     
         4 . A method according to  claim 3 , wherein the one or more macrocycles are one or more of cyclodextrins, calixarenes, cyclic peptides, crown ethers, cucurbiturils, pillararenes, derivatives thereof or a combination thereof. 
     
     
         5 . A method according to any one of the preceding claims, wherein the one or more molecular brakes are not one or more single stranded binding proteins (SSB). 
     
     
         6 . A method according to any one of the preceding claims, wherein the one or more molecular brakes are derived from one or more polynucleotide handling enzymes. 
     
     
         7 . A method according to  claim 6 , wherein the one or more polynucleotide handling enzymes are one or more polymerases, exonucleases, helicases, topoisomerases or a combination thereof. 
     
     
         8 . A method according to any one of the preceding claims, wherein the one or more molecular brakes are derived from one or more helicases. 
     
     
         9 . A method according to  claim 8 , wherein the one or more molecular brakes derived from 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. 
     
     
         10 . A method according to  claim 8  or  9 , wherein the one or more helicases and the one or more molecular brakes derived from helicases are not attached to one another except via the polynucleotide. 
     
     
         11 . A method according to any one of  claims 8  to  10 , wherein the one or more helicases and the one or more molecular brakes derived from helicases are different from one another. 
     
     
         12 . A method according to any one of  claims 8  to  11 , wherein the one or more molecular brakes derived from helicases are modified such that they bind the polynucleotide but do not function as a helicase. 
     
     
         13 . A method according to any one of  claims 8  to  21 , wherein the one or more molecular brakes derived from helicases are not stalled at a spacer. 
     
     
         14 . A method according to any one of the preceding claims, wherein the polynucleotide is a double stranded polynucleotide. 
     
     
         15 . A method according to  claim 14 , wherein in step (a) the one or more helicases are attached to a Y adaptor attached to one end of the double stranded polynucleotide and wherein the one or more molecular brakes are attached to a bridging moiety adaptor or hairpin loop adaptor attached to the other end of the double stranded polynucleotide. 
     
     
         16 . A method according to  claim 15 , wherein the one or more helicases and the one or more molecular brakes are brought together when the one or more helicases reach the bridging moiety or hairpin loop. 
     
     
         17 . A method according to  claim 15  or  16 , wherein the Y adaptor comprises a leader sequence which preferentially threads into the pore. 
     
     
         18 . A method according to any one of the preceding claims, wherein the polynucleotide is coupled to the membrane using one or more anchors. 
     
     
         19 . A method according to any one of the preceding claims, wherein in step (a) the one or more helicases are stalled at one or more spacers. 
     
     
         20 . A method according to  claim 19 , wherein the one or more spacers have a different structure from the polynucleotide. 
     
     
         21 . A method according to  claim 19  or  20 , wherein the one or more spacers comprise:
 (a) 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 or 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; 
 (b) one or more abasic nucleotides; and 
 (c) one or more chemical groups which cause the one or more helicases to stall. 
 
     
     
         22 . A method according to any one of the preceding claims, wherein the one or more helicases and the one or more molecular brakes control the movement of the polynucleotide through the pore with the field resulting from the applied potential. 
     
     
         23 . A method according to any one of the preceding claims, 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). 
     
     
         24 . A method according to any one of the preceding claims, wherein the pore is a transmembrane protein pore or a solid state pore. 
     
     
         25 . A method according to  claim 24 , 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. 
     
     
         26 . A method according to  claim 25 , wherein the transmembrane protein pore is:
 (a) MspA formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) a variant thereof in which one or more of the eight subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity; or   (b) α-hemolysin formed of seven identical subunits as shown in SEQ ID NO: 4 or (d) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity.   
     
     
         27 . A method of characterising a target polynucleotide, comprising:
 (a) carrying out the method of any one of the preceding claims; 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.   
     
     
         28 . A method according to  claim 27 , 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. 
     
     
         29 . A method according to  claim 28 , 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. 
     
     
         30 . A method according to any one of  claims 27  to  29 , wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement. 
     
     
         31 . A method according to  claim 30 , wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement. 
     
     
         32 . A kit for controlling the movement of a polynucleotide through a transmembrane pore, wherein the kit comprises one or more helicases and one or more molecular brakes. 
     
     
         33 . A kit according to  claim 32 , wherein the one or more molecular brakes are as defined in any one of  claims 2  to  13 . 
     
     
         34 . A kit according to  claim 32  or  33 , wherein the kit further comprises one or more anchors for coupling the polynucleotide to the membrane. 
     
     
         35 . A kit according to any one of  claims 31  to  34 , wherein the kit is for controlling the movement of a double stranded polynucleotide through a transmembrane pore and wherein the kit comprises a Y adaptor having one or more helicases attached and a bridging moiety adaptor or a hairpin loop adaptor having one or more molecular brakes attached. 
     
     
         36 . A kit according to  claim 35 , wherein the Y adaptor comprises a first anchor for coupling the polynucleotide to the membrane, the bridging moiety adaptor or the hairpin loop adaptor comprises a second anchor for coupling the polynucleotide to the membrane and wherein the strength of coupling of the second anchor to the membrane is greater than the strength of coupling of the first anchor to the membrane. 
     
     
         37 . A series of one or more helicases and one or more molecular brakes attached to a polynucleotide.