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The diverse functional roles of RNA are determined by its underlying structure. Accurate and comprehensive knowledge of RNA structure would inform a broader understanding of RNA biology and facilitate exploiting RNA as a biotechnological tool and therapeutic target. Determining the pattern of base pairing, or secondary structure, of RNA is a first step in these endeavors. Advances in experimental,...
Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA–protein interaction networks...
The structure determination of complex RNA molecules such as ribozymes, riboswitches and aptamers by X-ray crystallography hinges on the preparation of well-ordered crystals. Success usually results from molecular engineering to facilitate crystallization. An approach that has resulted in 10 new RNA structures in the past decade is the use of the U1A crystallization module. In this approach, the cognate...
Structured functional RNAs are conserved on the level of secondary and tertiary structure, rather than at sequence level, and so traditional sequence-based searches often fail to identify them. Structure-based searches are increasingly used to discover known RNA motifs in sequence databases. We describe the application of the program RNABOB, which performs such searches by allowing the user to define...
Lanthanide ions such as Tb 3+ and Eu 3+ have long been used to probe RNA and protein structures due to their luminescence properties and their steric and chemical similarities to biological metal ions such as Mg 2+ and Ca 2+ . In this article, we introduce a method that utilizes the enhanced Tb 3+ luminescence upon site-binding to RNA molecules as a FRET donor...
Among the greatest advances in biology today are the discoveries of various roles played by RNA in biological processes. However, despite significant advances in RNA structure determination using X-ray crystallography [1] and solution NMR [2–4], the number of bona fide RNA structures is very limited, in comparison with the growing number of known functional RNAs. This is because of great difficulty...
The crystallographic phase problem is the primary bottleneck encountered when attempting to solve macromolecular structures for which no close crystallographic structural homologues are known. Typically, isomorphous “heavy-atom” replacement and/or anomalous dispersion methods must be used in such cases to obtain experimentally-determined phases. Even three-dimensional NMR structures of the same macromolecule...
Charge flow and quenching (“CFQ”) is a relatively new, versatile, and easily carried out methodology for probing a number of unique features of DNA and RNA folded structures, and of their folding pathways. An electrical charge (an electron hole, or radical cation) is injected site-specifically into the end of a pre-determined reference helix within the larger DNA or RNA structure. The fate of the...
Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time...
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