Enzymes and systems for synthesizing DNA
AssigneeIridia, Inc.
InventorsPaul F. Predki, Stefen Boehme
Life of this patent
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Abstract
The disclosure provides novel topoisomerases which exhibit weakened ability to form a covalent bond to the 5′-(C/T)CCTT-3′ recognition site in the presence of increased NaCl concentrations relative to wild-type, together with novel methods for synthesizing DNA in the 3′ to 5′ direction using the novel topoisomerase; and other compounds, compositions, methods and devices comprising or utilizing topoisomerases which exhibit reduced covalent bond formation to the 5′-(C/T)CCTT-3′ recognition site in the presence of increased NaCl concentrations, relative to wild-type.
Claims4
A method of synthesizing a DNA molecule using topoisomerase-mediated ligation, by adding single nucleotides or oligomers to a DNA strand in the 3′ to 5′ direction, comprising (i) reacting a DNA molecule with a M-Topoisomerase charged with the desired nucleotide or oligomer wherein the nucleotide or oligomer is blocked from further addition at the 5′ end, then (ii) deblocking the 5′ end of the DNA thus formed, and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained, wherein the M-Topoisomerase is a topoisomerase comprising the following sequence:
- SEQ ID NO:
- 3
- RALFYKDGKLFTDNNFLNPVSDDNPAYEVLQHVKIPTHLTDVVVYEQTWEEALT RLIFVGSDSKGX1RX2X3FYGKMHVQNX4NAKRDRIFVRVYNVMKRINCFINKNIK KSSTDSNYQLAVFMLMETMFFIRFGKMKYLKENETVGLLTLKNKHIEISPDEIVIK FVGKDKVSHEFVVHKSNRLYKPLLKLTDDSSPEEFLFNKLSERKVYECIKQFGIRI KDLRTYGVNYTFLYNFWTNVKSISPLPSPKKLIALTIKQTAEVVGHTPSISKRAY MATTILEMVKDKNFLDVVSKTTFDEFLSIVVDHVKSSTDG
- wherein at least two of X1, X2, X3, and X4, are mutated from the residues in the wild type sequence [wherein in the wild-type sequence, X1 is arginine (R), X2 is glutamine (Q), X3 is tyrosine (Y), and X4 is arginine (R)] to glycine (G), alanine (A) or valine (V);
- provided that where only two residues are mutated, X1 and X2 are not both mutated to A.
A method for synthesizing DNA comprising reacting a donor DNA comprising a M-Topoisomerase bound to the 3′ phosphate of a terminal 5′-CCCTT-3′ sequence of the donor DNA with an acceptor DNA, so that the donor and acceptor DNA are ligated thereby and the M-topoisomerase is released, then dephosphorylating the 5′ terminal of the ligated DNA using a phosphatase, reacting the dephosphorylated DNA with a further donor DNA sequence comprising a M-Topoisomerase bound to the 3′ phosphate of a terminal 5′-CCCTT-3′ sequence, to obtain a further ligated DNA, and repeating until the desired sequence is obtained;
- wherein the M-Topoisomerase exhibits weakened covalent binding to the CCCTT recognition site in the presence of NaCl concentrations of greater than 200 mM, relative to a wild-type topoisomerase, and wherein the reaction takes place in a medium wherein the NaCl concentration is greater than 200 mM, wherein the M-Topoisomerase is a topoisomerase comprising the following sequence:
- SEQ ID NO:
- 3
- RALFYKDGKLFTDNNFLNPVSDDNPAYEVLQHVKIPTHLTDVVVYEQTWEEALTRLIFV GSDSKGX1RX2X3FYGKMHVQNX4NAKRDRIFVRVYNVMKRINCFINKNIKKSSTDSNYQ LAVFMLMETMFFIRFGKMKYLKENETVGLLTLKNKHIEISPDEIVIKFVGKDKVSHEFVV HKSNRLYKPLLKLTDDSSPEEFLFNKLSERKVYECIKQFGIRIKDLRTYGVNYTFLYNFW TNVKSISPLPSPKKLIALTIKQTAEVVGHTPSISKRAYMATTILEMVKDKNFLDVVSKTTF DEFLSIVVDHVKSSTDG
- wherein at least two of X1, and X4, are mutated from the residues in the wild type sequence [wherein in the wild-type sequence, X1 is arginine (R), X2 is glutamine (Q), X3 is tyrosine (Y), and X4 is arginine (R)] to glycine (G), alanine (A) or valine (V);
- provided that where only two residues are mutated, X1 and X2 are not both mutated to A.
Dependencies are inferred from the claim text.