Modified helicases
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
1. A method of controlling the movement of a polynucleotide with respect to a transmembrane pore, comprising
contacting the polynucleotide with a DNA-dependent ATPase (Dda) helicase modified to comprise a first cysteine residue and/or a first non-natural amino acid that has been introduced into (i) the tower domain, (ii) the pin domain, or (iii) the 1A (RecA-like motor) domain, wherein the first cysteine residue and/or the first non-natural amino acid is connected to a second cysteine residue and/or second non-natural amino acid that has been introduced into (i) the tower domain, (ii) the pin domain, or (iii) the 1A (RecA-like motor) domain and wherein the helicase retains its ability to control the movement of a polynucleotide with respect to the transmembrane pore, and thereby controlling the movement of the polynucleotide, in order to form a complex; and
contacting a transmembrane pore with the complex.
2. The method of claim 1 further comprising 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 target polynucleotide and thereby characterising the target polynucleotide.
3. The method according to claim 2 , wherein
the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.
4. The method according to claim 1 , wherein:
(A) the transmembrane pore is a transmembrane protein pore derived from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA transmembrane protein pore; or
(B) the transmembrane pore is:
(a) 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
(c) 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; or
(C) the transmembrane pore is a solid state pore.
5. The method of claim 1 , wherein the complex comprises an additional polynucleotide binding moiety, wherein:
the Dda helicase is attached to the additional polynucleotide binding moiety.