Enzymatic combinatorial nucleoside deletion scanning mutagenesis of functional RNA

Abstract

We describe a general and simple method to identify catalytically and structurally important nucleotides in functional RNAs. Our approach is based on statistical replacement of each nucleoside with a non-nucleosidic spacer (C3 linker, D), followed by separation of active library variants and readout of interference effects by analysis of enzymatic primer extension reactions.Many examples of natural and artificial functional RNA motifs have been identified, including ribozymes, aptamers, riboswitches, small interfering RNAs, or protein-binding RNAs, that demonstrate the diversity of RNA functions beyond the transmission of genetic information. 1 Elucidating the structure and sequence requirements of non-coding nucleic acids is crucial for understanding their mechanisms of action. Furthermore, such knowledge facilitates design and engineering of RNA for practical applications in biomolecular chemistry and synthetic biology.Numerous biochemical and biophysical methods are used to analyse nucleic acid architectures at various levels of resolution. 2 Recent additions to the traditional repertoire are geared towards high-throughput analyses on massively parallel arrays, or combine mutations and footprinting studies with computational methods. 3,4 Our laboratory has recently demonstrated combinatorial analysis methods (CoMA, 5 dNAIM, 6 NDS 7 ) that allowed the simultaneous assessment of all possible single point mutants of deoxyribozymes, as well as the identification of catalytically important nucleotides and their functional groups. However, due to the method design, the reported approaches are only applicable for studying functional DNAs. They are based on the combinatorial solid-phase synthesis of DNA oligonucleotide libraries, in which the mutations are marked with a chemical tag: the hydroxyl group resembling the 2 0 -OH group of ribonucleotides. Mutations tagged in this way can be easily decoded by alkaline hydrolysis. This is an asset for the analysis of functional DNAs, but, for obvious reasons, the same strategy cannot be applied to RNA.In the present work we established an analogously versatile combinatorial approach for RNA that reveals functionally important nucleotides in a simple set of experiments that do not require sophisticated instrumentation. We used the threecarbon linker D as non-nucleosidic spacer unit to statistically replace (''delete'') standard ribonucleotides within functional nucleic acids (Fig. 1). Following separation of the functionally active and inactive library variants, the spacer substitutions were decoded by primer extension reactions. Analysis of the primer extension pattern allowed identification of the essential and non-essential nucleotides. We demonstrated our new approach

On the mechanism of RNA phosphodiester backbone cleavage in the absence of solvent

“…Nucleoside phosphoramidites were obtained from ChemGenes (Wilmington, MA, USA) or Glen Research (Sterling, VA, USA). The propane-1,3-diol building block was prepared according to ( 45 , 46 ).…”

High definition polyphosphoesters: between nucleic acids and plastics

“…By creating a set of strands in which each of the nucleosides are individually replaced by C3, it is possible to identify which monomers are critical to enzymatic activity of catalytic RNAs. 52 Spacers also play an important part in DNA nanotechnology. In an early example, mPh3 units were used to create corners in DNA polygons 53 which could then be used to assemble nanotubes by stacking them up.…”