Abstract: The invention relates to substrates for O6-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X—CH2—R3—R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group —R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate, or a further group —R3—CH2—X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.

Abstract: The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against O6-alkylguanine substrates; (i) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted O6-alkylguanine substrates.

Abstract: The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against O6-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted O6-alkylguanine substrates.

Abstract: The invention relates to substrates for O6-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X—CH2—R3—R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group —R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate, or a further group —R3 CH2—X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.

Abstract: The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against 06-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted 06-alkylguanine substrates.