In vitro selection using a dual RNA library that allows primerless selection

Abstract

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C.

Selection‐by‐function: efficient enrichment of cathepsin E inhibitors from a DNA library

“…The difference in melting temperature between AT-rich and GC-rich (DT i ) was found to be well correlated with the experimental value obtained from mTGGE profile (Nishigaki et al, 2000). a significant extent in the recently introduced Tailored-SELEX that finally contains only $20% of the priming sequence (Vater et al, 2003;Jarosch et al, 2006).…”

Current Advances and Prospects on Implementation of Highly Sensitive Aptamer-based Dual System for Melamine Detection: New Promising Tool of Great Affinity

“…It has been designed an in vitro selection novel type of dual RNA library for mirror-image peptides detection like Ghrelin. It carries fixed sequences which constrains the oligonucleotides into a partially double-stranded structure that allows primer less selection in order to minimize the primer binding sequences to be part of the target-binding motif [56]. Moreover, DNA aptamers with only natural bases can often lack the desired specificity and binding affinity to target proteins compared to aptamers containing recombinant bases.…”

RNA Selection and EvolutionIn Vitro:Powerful Techniques for the Analysis and Identification of new Molecular Tools

“…This process, known as Tailored-SELEX (tailored systematic evolution of ligands by exponential enrichment), was used to identify oligonucleotides with affi nity for calcitonin generelated peptide. Three years later, this method was improved to be used with a conventional full-length library and a short library without PBSs (46). However, the incorporation of additional steps increased the complexity of the selection procedure, cancelling out the advantages of the technique.…”