Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins.

Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5'-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase.

2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.

2-Formyl-2'-deoxyadenosine triphosphate (d ATP) 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 d ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The d ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX).

Reactive modifications of DNA nucleobases for labelling, bioconjugations, and cross-linking.

Modification of DNA with reactive groups and their post-synthetic transformations are useful for labelling, imaging, bioconjugations and cross-linking with other (bio)molecules. This review summarizes the recent progress in this field and covers transformations of oxo groups, cycloadditions, conjugate additions, alkylations, cross-couplings and other reactions. Examples of applications are given and the practicability and scope of the reactions are discussed.

Squaramate-Modified Nucleotides and DNA for Specific Cross-Linking with Lysine-Containing Peptides and Proteins.

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.