RESUMO
This study investigated oligonucleotide (ON) synthesis containing 4'-selenoribonucleoside(s) under standard phosphoramidite conditions. Careful operation using a manual ON synthetic system revealed that an unexpected strand break occurred to afford a C2-symmetric homodimer as a byproduct. In addition, this side reaction occurred during I2 oxidation. On the basis of these findings, the first synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA was achieved using tert-butyl hydroperoxide (TBHP) as the alternative oxidant.
Assuntos
Oligonucleotídeos/síntese química , Compostos Organometálicos/síntese química , RNA/química , Selênio/química , terc-Butil Hidroperóxido/química , Estrutura Molecular , Oligonucleotídeos/química , Compostos Organometálicos/química , Oxidantes/química , OxirreduçãoRESUMO
The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).
Assuntos
Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/síntese química , Evolução Molecular Direcionada , Oligorribonucleotídeos/química , Tionucleotídeos/síntese química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntese química , Sequência de Bases , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , DNA Complementar/química , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Dados de Sequência Molecular , Oligorribonucleotídeos/metabolismo , Ribonuclease Pancreático/metabolismo , Tionucleotídeos/química , Tionucleotídeos/metabolismo , Trombina/metabolismo , Transcrição Gênica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virais/metabolismoRESUMO
We synthesized 4'-thioUTP (1) and 4'-thioCTP (2) with the aim of developing new NTP analogs for in vitro selection. Since in vitro selection requires both in vitro transcription and reverse transcription, we examined the ability of 1 and 2 for in vitro selection by focusing on both steps. Incorporation of 1 and 2 by T7 RNA polymerase to give 4'-thioRNA proceeded well and was superior to those of the two sets of frequently used modified NTP analogs (2'-NH2dUTP and 2'-NH2dCTP, and 2'-FdUTP and 2'-FdCTP) for in vitro selection. In addition, reverse transcription of the resulting 4'-thioRNA into the complementary DNA in the presence of dNTPs also proceeded smoothly and precisely. With these successful results in hand, in vitro selection of human a-thrombin RNA aptamer using 1 and 2 is in progress.