Investigations toward the selection of fully-modified 4'-thioRNA aptamers: optimization of in vitro transcription steps in the presence of 4'-thioNTPs.
We describe herein a method for isolating fully-modified 4'-thioRNA aptamers against human alpha-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4'-thioribonucleoside triphosphates (4'-thioNTPs) in an effort to afford the fully-modified 4'-thioRNA. The transcription efficiency of the 4'-thioNTPs was first compared with that of the nucleoside 5'-(alpha-thio)triphosphates (alphaSNTPs) and found to be less effective than that of the alphaSNTPs, especially when GTP and/or ATP were substituted for 4'-thioGTP and/or 4'-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4'-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4'-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4'-thioRNA, but not a fully-modified 4'-thioRNA, we eventually succeeded in isolating the fully-modified 4'-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers.
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. noriaki@pharm.hokudai.ac.jp
Noriaki
N
Minakawa
Mioko
M
Sanji
Yuka
Y
Kato
Akira
A
Matsuda
D000255:Adenosine Triphosphate; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics*; D052156:SELEX Aptamer Technique; D012689:Sequence Homology, Nucleic Acid; D013873:Thionucleotides / Q000235:genetics*; D013917:Thrombin / Q000235:genetics*; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
C501049:4'-thiocytidine 5-triphosphate; C501084:4'-thiouridine 5'-triphosphate; D052157:Aptamers, Nucleotide; D013873:Thionucleotides; D012313:RNA; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013917:Thrombin; D014544:Uridine Triphosphate