Isolation and characterization of fluorescence-enhancing RNA tags

An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries

“…Split-and-mix synthesis of one-bead one-compound libraries ensures that each physical entity (bead) contains multiple copies of one sequence. The Tentagel resins in combination with the strategy for sequence determination used here allow for large library sizes; 500 mg of 10 µm resin contain 10 9 beads, permitting complete randomization of 15 nt (4 15  ≈ 10 9 ), compared to 10 6 beads or 10 randomized nucleotides reported previously (13). Considering that 13- and 14-nt aptamers are known to bind small organic molecules with low micromolar affinity (34,35) and that a simple pentanucleotide accelerates an aminoacylation reaction 25-fold (36), OBOC RNA libraries such as the one described here should allow searching for a wide variety of functional oligonucleotides.…”

“…Furthermore, primer binding sites have often been found to be involved in target recognition (17). In the two examples cited (12,13), the constant regions were even larger in size than the variable ones. Whereas various approaches have been proposed to shorten or remove primer binding sites in the context of SELEX (16,18–21), there is still a need for complementary methods using on-bead libraries that do not rely on extended constant regions.…”

“…These authors created on-bead DNA libraries in which specific phosphodiester groups were replaced by phosphorothioates and phosphorodithioates, and isolated an efficient aptamer against a transcription factor (12). Recently, our group reported the isolation of a fluorescence-enhancing RNA tag out of an on-bead OBOC RNA library (13). In addition to the inclusion of chemical modifications, the advantage of on-bead screening in comparison to SELEX lies in the possibility for direct read-out: while SELEX selects only for binding strength or catalytic attachment or detachment to/from a matrix, on-bead screening can directly report measurable physical properties like a fluorescence, luminescence or polarization signal.…”

Combinatorial Chemistry Online

“…The RNA-derivatised beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement. 14 A simple and rapid method for the construction of phosphonic peptide mimetic inhibitor libraries -products of Ugi and Passerini multicomponent condensations -leading to the selection of new biologically active phosphonic pseudopeptides has been reported. The synthesised inhibitors were designed to target human neutrophil elastase, a serine protease whose uncontrolled activity may lead to development of several pathophysiological states such as rheumatoid arthritis, cystic fibrosis or tumour growth and invasion.…”

High‐Throughput Bead‐Based Identification of Structure‐Switching Aptamer Beacons

“…Bead-based RNA libraries and FACS have previously been used to identify RNA molecules capable of binding the small molecule dye FlAsH and enhancing its fluorescence [18] . In that report, the RNA libraries were generated chemically on beads using a ‘traditional’ split pool approach which prohibited performing multiple rounds of selection.…”