Hana Cahová

Aminophenyl‐ and Nitrophenyl‐Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection

DNA biosensors and chips are broadly utilized in the life sciences. Electrochemical detection is a less expensive but comparatively sensitive alternative to common optical methods. Although nucleic acids are electroactive themselves, diverse electroactive tags are used to increase sensitivity and specificity. Besides widely used DNA tags based on metal complexes, quantum dots, or phenothiazine dyes, some simple organic derivatives (such as aromatic amines or nitro compounds) exhibit distinct electrochemical activity, thus making them candidates for nucleic acid labeling. In particular, the nitro group appears promising for sensitive detection because of the high number of electrons (four or six) collected per nitro group reduction. So far, neither amino nor nitro groups have been used as specific electroactive DNA markers. Some modified 2’-deoxyribonucleoside triphosphates (dNTPs) bearing substituents at the nucleobase can be enzymatically incorporated into DNA by polymerases. This approach has been used for the construction of functionalized nucleic acids bearing diverse functional groups. Recently, aqueous-phase cross-coupling reactions of unprotected halogenated nucleoside triphosphates with boronic acids or acetylenes were developed and used in combination with polymerase incorporation for the two-step construction of modified nucleic acids, including ferrocene-labeled oligonucleotides (ONs). Herein, we report the synthesis of nucleoside triphosphates bearing aminophenyl and nitrophenyl groups attached to a nucleobase, their enzymatic incorporation, and the preliminary electrochemical properties of the labeled ONs. We expected that the fully conjugated aromatic system would respond well to the electronic changes arising from incorporation into nucleic acids, and result in changes of the redox potential of the label. The modified dNTPs were prepared by the single-step aqueous-phase cross-coupling reactions of halogenated dNTPs, in analogy to our previously developed procedures. The Suzuki–Miyaura reaction of 7-iodo-7-deaza-2’-deoxyadenosine 5’-triphosphate (7-I-7-deaza-dATP), 5-iodo-2’deoxyuridine 5’-triphosphate (5-I-dUTP), or 5-iodo-2’-deoxycytidine 5’-triphosphate (5-I-dCTP) with either 3-aminophenylor 3-nitrophenylboronic acid (Scheme 1) gave the

Turning Off Transcription with Bacterial RNA Polymerase through CuAAC Click Reactions of DNA Containing 5‐Ethynyluracil

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (UE ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA template containing UE was used for in vitro transcription with E. coli RNA polymerase and the transcription was turned off by CuAAC with 3-azidopropane-1,2-diol or 3-azido-7-hydroxycoumarin.

Polymerase Synthesis of Base-Modified DNA

Enzymatic synthesis of base-modified DNA by polymerase incorporation of modified nucleotides is discussed. Modified 2′-deoxyribonucleoside triphosphates (dNTPs) are key substrates for polymerases and can be prepared either by triphosphorylation of modified nucleosides or by direct aqueous cross-coupling reactions of halogenated dNTPs with alkynes, arylboronic acids, or alkenes. The methods of polymerase synthesis include primer extension, PCR, nicking enzyme amplifications, and other methods which enable the synthesis of diverse types of long or short and double-stranded DNA or single-stranded oligonucleotides. The applications include labeling in diagnostics (labeling or coding of DNA bases) and chemical biology (bioconjugations, modulation of protein binding, etc.).

Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications

A novel efficient two-step methodology for the construction of base-functionalized DNA is based on direct aqueous cross-coupling reactions of unprotected nucleoside triphosphates followed by polymerase incorporation. Preliminary applications of the modified DNA in electrochemical detection and bioanalysis are outlined.