Selection and characterization of RNA aptamers that bind fluorogenic cyanine dyes

RNA molecules are involved in many essential processes in the cellular methabolism. In addition to the well-understood roles in information transfer and protein synthesis, RNA is also involved in splicing, gene regulation, maturation of other cellular RNAs and telomere maintenance and (likely) other, as yet unknown cellular processes.;RNA-detection tools are needed for the study and understanding of the cellular RNAs. We developed fluorescent RNA tags composed of RNA aptamers that specifically bind the fluorogenic cyanine dye DIR and activate its fluorescence. We designed DIR to display low affinity for nucleic acids, then we selected RNA aptamers that bind DIR specifically with nanomolar affinity. The fluorescence of DIR is significantly increased upon binding to the specific aptamers.;We selected RNA aptmamers for DIR from two different starting pools, each of which contains a stem-loop element flanked by two randomized regions. The first pool was symmetric with the stem-loop in the center, the second loop was asymmetric with the stem-loop shifted towards the end of the randomized region. The best aptamers selected from each pool increase the DIR fluorescence to similar levels, but they differ in their binding affinity. The aptamer from the symmetric pool is poorly represented in the final pool of winners. The aptamer from the asymmetric pool becomes very abundant in the pool of winners at the end of the selection. A truncation of this aptamer that is only 49 nucleotides in length and still retains function was identified.;The shortest aptamer that is still able to increase the DIR fluorescence was transcribed in vitro in the presence of DIR. It was observed that it is possible to monitor the transcription process in real time by measuring the fluorescence intensity of DIR as a function of time. This observation opens up the possibility of using such RNA aptamers as fluorescent RNA tags for in vivo RNA monitoring.;In order to further understand the binding between RNA aptamers and DIR, the binding constants between various aptamers and 5 DIR-related cyanine dyes were measured. The 5 cyanine dyes show comparable affinities for most tested aptamers, suggesting that side-chain modifications on DIR could be tolerated by the aptamers. Such side-chain modifications could be used to improve dye properties such as membrane permeability.