Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways

This overview unit discusses fluorescence spectroscopy as a tool for studying RNA folding. Ribozymes and oligonucleotides can be labeled with a fluorescent probe and analyzed to give information about both slow and fast kinetic processes with real‐time data acquisition. The unit discusses the advantages and disadvantages of various pendant probes and nucleotide analogs, the analytical methods that can be used, instrument setup, control experiments, and a variety of kinetic experiments that can be performed, such as determination of rate constants.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143791/1/cpnc1108.pd

RNA pseudoknots: folding and finding

RNA pseudoknots are important for function. Three-dimensional structural information is available, insights into factors affecting pseudoknot stability are being reported, and computer programs are available for predicting pseudoknots

A CA+ Pair Adjacent to a Sheared GA or AA Pair Stabilizes Size-Symmetric RNA Internal Loops†

ABSTRACT: RNA internal loops are often important sites for folding and function. Residues in internal loops can have pKa values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the CG CA AA and AA nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 3 nucleotide and larger size-symmetric internal loops. These CG C

The influence of locked nucleic acid residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA

The Membership and Distance of the Open Cluster Collinder 419

The young open cluster Collinder 419 surrounds the massive O star, HD 193322, that is itself a remarkable multiple star system containing at least four components. Here we present a discussion of the cluster distance based upon new spectral classifications of the brighter members, UBV photometry, and an analysis of astrometric and photometric data from the UCAC3 and 2MASS catalogs. We determine an average cluster reddening of E(B-V)=0.37 +- 0.05 mag and a cluster distance of 741 +- 36 pc. The cluster probably contains some very young stars that may include a reddened M3 III star, IRAS~20161+4035

Conformational ensembles of RNA oligonucleotides from integrating NMR and molecular simulations

RNA molecules are key players in numerous cellular processes and are characterized by a complex relationship between structure, dynamics, and function. Despite their apparent simplicity, RNA oligonucleotides are very flexible molecules, and understanding their internal dynamics is particularly challenging using experimental data alone. We show how to reconstruct the conformational ensemble of four RNA tetranucleotides by combining atomistic molecular dynamics simulationswith nuclear magnetic resonance spectroscopy data. The goal is achieved by reweighting simulations using a maximum entropy/Bayesian approach. In this way, we overcome problems of current simulation methods, as well as in interpreting ensemble- and time-averaged experimental data. We determine the populations of different conformational states by considering several nuclear magnetic resonance parameters and point toward properties that are not captured by state-of-the-art molecular force fields. Although our approach is applied on a set of model systems, it is fully general and may be used to study the conformational dynamics of flexible biomolecules and to detect inaccuracies in molecular dynamics force fields