In vitro selection of human alpha-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP.
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.
Kato
Yuka
Y
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Minakawa
Noriaki
N
Ogawa
Naoki
N
Matsuda
Akira
A
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D052156:SELEX Aptamer Technique / Q000379:methods*; D013997:Time Factors; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry
C501049:4'-thiocytidine 5-triphosphate; C501084:4'-thiouridine 5'-triphosphate; D052157:Aptamers, Nucleotide; C081676:thrombin aptamer; D003570:Cytidine Triphosphate; D014544:Uridine Triphosphate