Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.
Abstract2‐Formyl‐2′‐deoxyadenosine triphosphate (dCHOATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHOATP was based on the preparation of protected 2‐dihydroxyethyl‐2′‐deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHOATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31‐mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single‐nucleotide extension followed by PEX was used for site‐specific incorporation of one aldehyde‐linked adenosine into a longer 49‐mer sequence. The reactive formyl group was used for cross‐linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647‐linked hydroxylamine.
Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.
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