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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
Published on: August 21, 2018
In vitro selection of human alpha-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP
Yuka Kato1, Noriaki Minakawa, Naoki Ogawa
1Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Abstract:
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.
