RESUMEN
In bioconjugation chemistry, achieving a target-specific reaction for a non-modified amino acid is challenging. Here, we report a novel nucleobase-involved native chemical ligation (NbCL) that allows a site-specific oligonucleotide-peptide conjugation via a new S-N acyl transfer reaction between an oxanine nucleobase and N-terminal cysteine.
Asunto(s)
Cisteína/química , Oligonucleótidos/química , Péptidos/química , Nucleósidos de Purina/química , Estructura MolecularRESUMEN
Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.
Asunto(s)
Boratos/química , ADN Nucleotidilexotransferasa/metabolismo , Oligodesoxirribonucleótidos/química , Ribonucleótidos/química , Compuestos Alílicos/química , Biotina/química , Tampones (Química) , ADN Nucleotidilexotransferasa/antagonistas & inhibidores , Oligodesoxirribonucleótidos/biosíntesis , Polimerizacion , Nucleósidos de Purina/químicaRESUMEN
A highly accurate ligase-based SNP analysis was developed by using modified base-end downstream ligation fragments as detection probes, which can clearly distinguish C/T SNP types without any "false-positive" results.