Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.

• MeSH
• azidy chemie   MeSH
• click chemie MeSH
• deoxyribonukleotidy chemická syntéza   chemie   metabolismus   MeSH
• DNA primery biosyntéza   genetika   MeSH
• endonukleasy metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• molekulární struktura MeSH
• oligonukleotidy biosyntéza   MeSH
• polymerázová řetězová reakce * MeSH
• techniky amplifikace nukleových kyselin * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• deoxyribonukleotidy MeSH
• DNA primery MeSH
• endonukleasy MeSH
• fluorescenční barviva MeSH
• oligonukleotidy MeSH

Commercially available trans-4-hydroxy-L-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with N-phosphonomethyl moiety attached to the nitrogen atom of prolinol ring. The synthetic methodology based on the inversion of configuration at both 1- and 4- positions led, in result, to all diastereoisomeric O-protected 4-mesyloxyprolinol-N-methylphosphonates. Alkylation of nucleobases using the synthons afforded the nucleotide analogues corresponding to alpha- and beta-nucleotides in both L- and D-series. The NMR-based conformational study of alpha- and beta-nucleotides in aqueous solution performed at two different pH values securing either N-fully protonated or deprotonated forms revealed in both cases occurrence of the same mostly populated conformer. All final prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found.

• MeSH
• deoxyribonukleotidy chemická syntéza   chemie   MeSH
• nukleosidy chemie   MeSH
• organofosfonáty chemická syntéza   chemie   MeSH
• pyrrolidiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleotidy MeSH
• nukleosidy MeSH
• organofosfonáty MeSH
• prolinol MeSH Prohlížeč
• pyrrolidiny MeSH