Modified helicases
The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.
1. A complex comprising (i) a monomeric helicase that comprises a polynucleotide binding domain and two RecA-like domains, and (ii) a target polynucleotide bound to the polynucleotide binding domain, wherein two amino acid residues that are located in different structural domains on the surface of the helicase surrounding the polynucleotide binding domain are artificially covalently connected via a linkage between the two amino acid residues, such that the helicase has a covalently-closed structure and the distance between the two amino acids is less than 50 Angstroms (Å), wherein the bound target polynucleotide is encircled by the covalently-closed structure.
2. The complex according to claim 1 , wherein at least one amino acid of the two amino acid residues is substituted with cysteine, a non-natural amino acid or 4-azido-L-phenylalanine (Faz).
3. The complex according to claim 1 , wherein the two amino acid residues are on one or more loop regions connecting α-helices and β-strands of the helicase and/or are spatially located proximal to the polynucleotide binding domain.
4. The complex according to claim 1 , wherein the helicase is (a) a Hel308 helicase, a RecD helicase, a TraI helicase, a TraI subgroup helicase, an XPD helicase or a variant thereof; or (b) one of the helicases as set forth in SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 29, 32-34, 37-55, 58, 85, 126, 134, 138 or a variant thereof.
5. The complex according to claim 1 , wherein the helicase further comprises a second set of two amino acid residues in different structural domains of the helicase surrounding the polynucleotide binding domain that are artificially covalently connected via a linkage between the two amino acid residues of the second set.
6. The complex according to claim 1 , wherein the linkage between the two amino acid residues comprises an amino acid sequence.
7. The complex according to claim 6 , wherein the amino acid sequence comprises a polynucleotide binding moiety.
8. The complex according to claim 1 , wherein the linkage between the two amino acid residues comprises a polyethyleneglycol (PEG), polysaccharide, or polyamide.
9. The complex according to claim 1 , wherein the linkage between the two amino acid residues comprises a deoxyribonucleic acid (DNA) sequence, peptide nucleic acid (PNA), threose nucleic acid (TNA), or glycerol nucleic acid (GNA).
10. The complex according to claim 1 , wherein the helicase is a member of a helicase family selected from the group consisting of: Pifl-like helicases, Upfl-like helicases, UvrD/Rep helicases, Ski-like helicases, Rad3/XPD helicases, NS3/NPH-II helicases, DEAD helicases, DEAHi RHA helicases, RecG-like helicases, REcQ-like helicases, T1R-like helicases, Swi/Snf-like helicases, and Rig-I-like helicases.
11. The complex according to claim 1 , wherein the helicase is a member of a helicase subfamily selected from the group consisting of: RecD helicases, Upfl helicases, PcrA helicases, Rep helicases, UvrD helicases, Hel308 helicases, Mtr4 helicases, XPD helicases, NS3 helicases, Mssl 16 helicases, Prp43 helicases, RecG helicases, RecQ helicases, T1R helicases, RapA helicases and Hef helicases.
12. The complex according to claim 1 , wherein the helicase has a covalently-closed structure and the distance between the two amino acids is less than 30 Å.