Generating New Specific Rna Interaction Interfaces Using C-loops

New RNA interaction interfaces are reported for designing RNA modules for directional supramolecular self-assembly. The new interfaces are generated from existing ones by inserting C-loops between the interaction motifs that mediate supramolecular assembly. C-Loops are new modular motifs recently identified in crystal structures that increase the helical twist of RNA helices in which they are inserted and thus reduce the distance between pairs of loop or loop-receptor motifs from 11 to 9 base-stacking layers while maintaining correct orientation for binding to cognate interaction interfaces. Binding specificities of C-loop-containing molecules for cognate molecules that also have inserted C-loops were found to range up to 20-fold. Binding affinities for most C-loop-containing molecules were generally equal or higher than those for the parent molecules lacking C-loops

Specific Rna Self-assembly With Minimal Paranemic Motifs

The paranemic crossover (PX) is a motif for assembling two nucleic acid molecules using Watson-Crick (WC) basepairing without unfolding preformed secondary structure in the individual molecules. Once formed, the paranemic assembly motif comprises adjacent parallel double helices that crossover at every possible point over the length of the motif. The interaction is reversible as it does not require denaturation of basepairs internal to each interacting molecular unit. Paranemic assembly has been demonstrated for DNA but not for RNA and only for motifs with four or more crossover points and lengths of five or more helical half-turns. Here we report the design of RNA molecules that paranemically assemble with the minimum number of two crossovers spanning the major groove to form paranemic motifs with a length of three half turns (3HT). Dissociation constants (K-d\u27s) were measured for a series of molecules in which the number of basepairs; between the crossover points was varied from five to eight basepairs. The paranemic 3HT complex with six basepairs (3HT_6M) was found to be the most stable with K-d = 1 x 10(-8) M. The half-time for kinetic exchange of the 3HT_6M complex was determined to be similar to 100 min, from which we calculated association and dissociation rate constants k(a) = 5.11 x 10(3) M(-1)s(-1) and k(d) = 5.11 x 10(-5) s(-1). RNA paranemic assembly of 3HT and 5HT complexes is blocked by single-base substitutions that disrupt individual intermolecular Watson-Crick basepairs; and is restored by compensatory substitutions that,restore those basepairs. The 3HT motif appears suitable for specific, programmable, and reversible tecto-RNA self-assembly for constructing artificial RNA molecular machines

Calculation of Splicing Potential from the Alternative Splicing Mutation Database

© 2008 Bechtel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Attenuation of loop-receptor interactions with pseudoknot formation

RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology

Design and Characterization of Novel Bio-Sensor Platform for Sequence Specific, Label-Free, Fluorescent Detection of Native RNA Moledcules

This project describes a new bio-sensor platform for sequence-specific, label-free,fluorescent detection of pre-folded RNA molecules. This detection is based on paranemic association of a sensor RNA molecule to a target RNA molecule using Watson-Crick basepairing. We demonstrate the paranemic association can be accomplished with a minimal 3-half-turn (3HT) paranemic pairing motif. The motif results from two strand exchanges between the sensor and target RNAs that allow for the formation of 10 to 16 inter-molecular Watson-Crick basepairs in major (M) groove or 8 to 14 inter-molecular Watson-Crick basepairs in minor (m) groove when the sensor and target molecules have complementary sequences. Paranemic association does not require unfolding of preformed secondary structures in either the sensor or target molecules. This poject teaches how to position and orient an aptamer for the triphenylmethane dye Malachite Green (MG) within the sensor RNA so that the sensor RNA only binds MG at the aptamers site when it is bound in turn to the target RNA. When the sensor/target complex forms, it binds MG at the aptamer site and the MG becomes fluorescent and thus signals the presence of the target RNA. In the absence of the target RNA, the sensor RNA is not able to bind MG, so the MG remains free in solution and no fluorescence is observed. Thus the system performs as a fluorescent sensor for the target RNA without the need to covalently attach a fluorescent moiety to either the sensor or the target. This fluorescent sensor system also has the potential to be used as an RNA-chromophore-assisted laser inactivation (RNA-CALI) agent providing light-induced degradation of the sensor/target complex. Also in this dissertation proposal we investigate how to modulate the helical twist of RNA molecules using C-loop, a new modular recurrent RNA motif, recently identified in crystal structures, to generate a new, specific self-assembly interface, using known cognate loop-receptor motifs. This design is intended for use in a second biosensor platform design that employs specific loop-receptor interactions and is still under development. Furthermore, these results shed new light on possible roles for these motifs in biological structures

Smart-Responsive Nucleic Acid Nanoparticles (NANPs) with the Potential to Modulate Immune Behavior

Nucleic acids are programmable and biocompatible polymers that have beneficial uses in nanotechnology with broad applications in biosensing and therapeutics. In some cases, however, the development of the latter has been impeded by the unknown immunostimulatory properties of nucleic acid-based materials, as well as a lack of functional dynamicity due to stagnant structural design. Recent research advancements have explored these obstacles in tandem via the assembly of three-dimensional, planar, and fibrous cognate nucleic acid-based nanoparticles, called NANPs, for the conditional activation of embedded and otherwise quiescent functions. Furthermore, a library of the most representative NANPs was extensively analyzed in human peripheral blood mononuclear cells (PBMCs), and the links between the programmable architectural and physicochemical parameters of NANPs and their immunomodulatory properties have been established. This overview will cover the recent development of design principles that allow for fine-tuning of both the physicochemical and immunostimulatory properties of dynamic NANPs and discuss the potential impacts of these novel strategies