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

Abstract

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.

Purine Nucleosides with a Reactive (β‐Iodovinyl)sulfone or a (β‐Keto)sulfone Group at the C8 Position and Their Polymerase‐Catalyzed Incorporation into DNA

“…Modified nucleosides received great attention because of their potential to function as anticancer and antiviral therapeutics. [1,2] Nucleosides with modified purine bases bearing reactive groups such as azide, [3,4] alkene, [5,6] alkyne, [6] aldehyde [7] or radical precursors [8][9][10] have been explored for imaging cellular DNA, bioconjugation with DNA-bound proteins, DNA damage repair, and applications in the fluorescent bioanalysis [2,11] of DNA and RNA. The 5' triphosphate or 3' phosphoroamidite of these probes were incorporated into oligonucleotides (ON) by DNA/ RNA polymerases [6,12,13] or solid-phase synthesis.…”

Mononucleotide repeat expansions with non-natural polymerase substrates

“…One of the major characteristics of non-natural dNTPs is their substrate properties with respect to DNA polymerases. The estimation of this parameter in the primer extension (PEX) reaction is a routine procedure in the characterization of newly synthesized modified dNTPs [6][7][8][9] . In particular, homopolymer DNA templates may be used for the estimation of the polymerization efficiency of non-natural nucleotides to verify the possibility of multiple consecutive incorporations 10,11 .…”

1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

“…[9] Vinylsulfonamide group was also used to cross-link with Cys through conjugate addition, [10] whereas chloroacetamide alkylated Cys or His in proteins. [11] Most commonly used was cross-linking of aldehyde-linked DNA with Lys either through reversible imine formation [12] or through irreversible reductive amination [13,14] that requires additional NaBH 3 CN as stoichiometric reductant. Recently we have reported squaramate-linked DNA that reacted with Lys-containing peptides and proteins without any additional reagent.…”