In Vitro Selection without Intervening Amplification

摘要

Aptamers to medically important targets have potential value as drug leads. Currently, aptamer selection procedures are typically limited to unmodified DNA and RNA. When modified oligomers are incorporated, the selection procedure is terminated afterjust one round of amplification since polymerases will not tolerate most chemically modified mononucleotides. However, if the modified nucleotides are not part of the random region that is amplified by the polymerase chain reaction (PCR) after each selection, they would not disturb the amplification. Unfortunately, using such a method would require reincorpora-tion of the modified oligomers into the library after each selection cycle, making the method quite tedious. This limitation would be circumvented if the screening process did not involve amplification during each selection cycle, but instead relied on a single amplification of the random region after the final round of selection. If the modified region was separated from the random region by a PCR primer (Scheme 1), then the successful aptamer could be resynthesized in a manner similar to that used to produce the original library. In addition, such a recursive selection without intervening amplification should yield high-affinity aptamers in afraction of the time required for in vitro selection. Although we do not describe the use of modified nucleotides here, we introduce three requirements for in vitro selection without intervening amplification and demonstrate its viability.