In vitro and in vivo production and purification of circular RNA aptamer.
RNA aptamers are potential candidates for RNA therapeutics. They must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. Since circular RNA molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin RNA aptamer both in vitro and in vivo. Circularization was accomplished by self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. This sequence was producible in both Escherichia coli cells and in vitro. The circularized streptavidin RNA aptamer retained its binding of streptavidin and was stabile in HeLa cell extracts compared to the linear form of the streptavidin aptamer. The self-spliced circular RNA from the transcribed permuted intron-exon transcripts in E. coli cells was purified from a total RNA fraction using the solid-phase DNA probe method following anion exchange chromatography that excluded gel electrophoresis. This study provides an alternative method for designing and purifying useful RNA aptamers.
Umekage
So
S
Department of Ecological Engineering, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi 441-8580, Japan. umekage@eco.tut.ac.jp
Kikuchi
Yo
Y
D052157:Aptamers, Nucleotide / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000079962:RNA, Circular; D044967:Serum; D019809:Streptavidin / Q000378:metabolism
D052157:Aptamers, Nucleotide; D012329:RNA, Bacterial; D000079962:RNA, Circular; D012313:RNA; D019809:Streptavidin