Accelerated, high-quality photolithographic synthesis of RNA microarrays in situ.

Abstract

Nucleic acid photolithography is the only microarray fabrication process that has demonstrated chemical versatility accommodating any type of nucleic acid. The current approach to RNA microarray synthesis requires long coupling and photolysis times and suffers from unavoidable degradation postsynthesis. In this study, we developed a series of RNA phosphoramidites with improved chemical and photochemical protection of the 2'- and 5'-OH functions. In so doing, we reduced the coupling time by more than half and the photolysis time by a factor of 4. Sequence libraries that would otherwise take over 6 hours to synthesize can now be prepared in half the time. Degradation is substantially lowered, and concomitantly, hybridization signals can reach over seven times those of the previous state of the art. Under those conditions, high-density RNA microarrays and RNA libraries can now be synthesized at greatly accelerated rates. We also synthesized fluorogenic RNA Mango aptamers on microarrays and investigated the effect of sequence mutations on their fluorogenic properties.