Direct Polymerase Synthesis of Reactive Aldehyde‐Functionalized DNA and Its Conjugation and Staining with Hydrazines

Abstract

Apart from a wide range of novel applications of functionalized DNA in chemical biology, nanotechnology, and material sciences, [1] attachment of reactive functional groups to nucleic acids is needed for further transformations or bioconjugates. The introduction of alkyne, azide, or diene groups either by chemical phosphoramidite synthesis or by enzymatic polymerase synthesis has been achieved and the modified DNA was used for click-chemistry, [2,3] Staudinger ligation, [4] and Diels-Alder reactions. [5] An aldehyde functional group is a very attractive group because of its high and specific reactivity with diverse reagents. However, it was considered too reactive and fragile to be incorporated directly (chemically or enzymatically) [6] and the few successful examples were prepared indirectly by a click reaction with azide derivatives of reducing sugars, [3] or by introduction of 2,3-dihydroxypropyl or 3,4-dihydroxypyrrolidine moieties [7,8] and subsequent oxidative cleavage of the vicinal diols to (di)aldehydes. The syntheses of the nucleoside/nucleotide monomers were laborious multistep procedures and additional post-synthetic steps were required to release the aldehyde function in DNA. [7,8] Metallization [7] or interstrand cross-linking [8] were demonstrated to be very useful applications of aldehyde-modified oligonucleotides (ONs) or DNA. Therefore we decided to develop a simple and efficient direct protocol for construction of aldehyde-modified DNA by application of our two-step (cross-coupling polymerase incorporation) method. [9,10] In addition, we wished to develop a methodology for additional conjugation and staining of aldehyde-modified DNA by hydrazone formation.The methodology of choice involved Suzuki cross-coupling of a halogenated nucleoside triphosphate (dNTP) with an aldehyde-containing boronic acid, and subsequent polymerase incorporation into DNA. [9,10] Furthermore, we wanted to develop a general methodology for hydrazone formation in aqueous media. To test the first and last steps of our proposed route, we performed the reactions on the model compound 5iodo-dCMP (1; dCMP = 2'-deoxycytidine-5'-O-monophos-phate). Commercially available 5-formylthiophene-2-boronic acid was selected as a suitable carrier for the aldehyde group, and its aqueous-phase cross-coupling with monophosphate 1 proceeded within 40 minutes and gave aldehyde-modified dCMP 2 in 50 % yield (Scheme 1). The next task was the formation of the hydrazone species, which is usually only performed in dry organic solvents (owing to the formation of water in the reaction). To make the reaction amenable to aqueous conditions, we have adapted the protocol developed by Dawson and co-workers [11] for aqueous conjugation of peptides, which uses aqueous ammonium acetate and aniline to facilitate the condensation. To test the reactions with 2, we selected two arylhydrazines (3 and 4) that are commonly used as aldehyde-specific dyes. [12,13] The reactions of aldehydenucleotide 2 with 3 or 4 proceeded at room temperature for approximately 20 ho...

7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

“…We selected a series of eight previously reported 7substituted 7-deaza-dNTPs and six 5-substituted dCTPs bearing various bulky functional groups. They included ethynyl and phenyl derivatives, [10] redox labels (dN NO2 TP and dN NH2 TP), [12] fluorescent labels (dA BFU TP, dA ABOX TP), [13] reactive groups (dC FT TP), [14] and very bulky groups (dN STr TP). [15] They were incorporated through competitive PEX by different DNA polymerases (Pwo, KOD XL, Taq, Vent(exo-), Klenow, Bst, and human Pol a) to different sequences in different ratios of modified dN X TP to unmodified dNTP (1:1 and 10:1).…”

“…They included ethynyl and phenyl derivatives, [10] redox labels (dN NO2 TP and dN NH2 TP), [12] fluorescent labels (dA BFU TP, dA ABOX TP), [13] reactive groups (dC FT TP), [14] and very bulky groups (dN STr TP). They included ethynyl and phenyl derivatives, [10] redox labels (dN NO2 TP and dN NH2 TP), [12] fluorescent labels (dA BFU TP, dA ABOX TP), [13] reactive groups (dC FT TP), [14] and very bulky groups (dN STr TP).…”

Vinylsulfonamide and Acrylamide Modification of DNA for Cross‐linking with Proteins

“…Introduction of some reactive groups, that is, alkyne, alkene, azide, diene, or aldehyde groups or a cysteine residue, was achieved either by chemical phosphoramidite synthesis or by enzymatic polymerase synthesis. The modified DNAs were used for [2+3]‐dipolar cycloadditions,3, 4 Staudinger ligation,5 Diels–Alder reactions,6 hydrazone formation,7 reductive aminations,8 or native peptide ligations 9. However, none of these reactive groups allow bioorthogonal and specific cross‐linking with a natural amino acid residue in a peptide or protein.…”

Transient and Switchable (Triethylsilyl)ethynyl Protection of DNA against Cleavage by Restriction Endonucleases

“…The polymerase construction of base‐modified DNA10 is now an established and widely used procedure. In our group, we combined this procedure with the aqueous cross‐coupling of halogenated dNTPs11 into an efficient two‐step synthesis of DNA bearing redox labels12 or reactive functional groups 13. Therefore, we first tried the aqueous Sonogashira cross‐coupling reactions of dA I TP with (trialkylsilyl)acetylenes (Scheme ).…”