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 5-iodo-dCMP (1; dCMP = 2'-deoxycytidine-5'-O-monophosphate). 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...

DNA-Multichromophore Systems

“…The PCR product can then be treated with arylhydrazines for bioconjugation and staining purposes. 262 …”

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA–Protein Cross‐Linking

“…[2] In particular, bioconjugation of nucleic acids with peptides, proteins, or lipids has found many applicationsi nm edicinal chemistry,c hemicalb iology,a nd nanotechnology. Recent advances in oligonucleotide synthesis (either chemicalp hosphoramidite [5] or enzymatics ynthesis by using base-modified 2'-deoxynucleosidet riphosphates [6] (dNTPs))h as enabledt he introductiono fm any reactive groups into DNA (e.g.,aldehyde, [7] alkyne, [8,9] alkene, [10] diene, [11] azide, [12] or tetrazole [13] ). Recent advances in oligonucleotide synthesis (either chemicalp hosphoramidite [5] or enzymatics ynthesis by using base-modified 2'-deoxynucleosidet riphosphates [6] (dNTPs))h as enabledt he introductiono fm any reactive groups into DNA (e.g.,aldehyde, [7] alkyne, [8,9] alkene, [10] diene, [11] azide, [12] or tetrazole [13] ).…”

Chemistry of nucleic acids: impacts in multiple fields

“…For example, metal-mediated carbon-carbon bond forming chemistries have shown real utility in recent years. 6 …”

“…12 Hocek has demonstrated methods for rapidly introducing aldehydes on DNA bases, which yields a ready site for addition of labels. 6c …”