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Synthetic RNA recognition motifs that selectively recognize HIV-1 trans-activation response element hairpin RNA.

ABSTRACT: A multitude of RNA hairpins are directly implicated in human disease. Many of these RNAs are potentially valuable targets for drug discovery and basic research. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. Although a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective RNA-binding molecules remains a daunting challenge. RNA recognition motif (RRM) domains may represent unique privileged scaffolds for the generation of synthetic proteins that selectively recognize structured disease-relevant RNAs, including RNA hairpins. As a demonstration of this potential, we mutated putative RNA-binding regions within the U1A RRM and a variant thereof and screened these synthetic proteins for affinity to HIV-1 trans-activation response (TAR) element hairpin RNA. Some of these U1A-derived proteins bind TAR with single-digit micromolar dissociation constants, and they do so preferentially over the native protein's original target RNA (U1hpII) and a DNA TAR variant. Binding affinity is not appreciably diminished by addition of 10 molar equivalents of cellular tRNAs from Escherichia coli. Taken together, our findings represent the first synthetic RRMs that selectively bind a disease-relevant RNA hairpin and may represent a general approach for achieving sequence-selective recognition of RNA hairpins, which are the focus of therapeutic discovery and basic research.

SUBMITTER: Blakeley BD

PROVIDER: S-EPMC4414246 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Synthetic RNA recognition motifs that selectively recognize HIV-1 trans-activation response element hairpin RNA.

Blakeley Brett D BD McNaughton Brian R BR

ACS chemical biology 20140325 6

A multitude of RNA hairpins are directly implicated in human disease. Many of these RNAs are potentially valuable targets for drug discovery and basic research. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. Although a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective RNA-binding molecules remains a ...[more]

PMID: 24635165

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Project description:The conformations and stabilities of the β-hairpin model peptides of Waters (Riemen, A. J.; Waters, M. L. Biochemistry 2009, 48, 1525; Hughes, R. M.; Benshoff, M. L.; Waters, M. L. Chemistry 2007, 13, 5753) have been experimentally characterized as a function of lysine ε-methylation. These models were developed to explore molecular recognition of known epigenetic recognition motifs. This system offered an opportunity to computationally examine the role of cation-π interactions, desolvation of the ε-methylated ammonium groups, and aromatic/aromatic interactions on the observed differences in NMR spectra. AMOEBA, a second-generation force field (Ponder, J. W.; Wu, C.; Ren, P.; Pande, V. S.; Chodera, J. D.; Schnieders, M. J.; Haque, I.; Mobley, D. L.; Lambrecht, D. S.; DiStasio, R. A., Jr.; Head-Gordon, M.; Clark, G. N.; Johnson, M. E.; Head-Gordon, T. J. Phys. Chem. B 2010, 114, 2549), was chosen as it includes both multipole electrostatics and polarizability thought to be essential to accurately characterize such interactions. Independent parametrization of ε-methylated amines was required from which aqueous solvation free energies were estimated and shown to agree with literature values. Molecular dynamics simulations (100 ns) using the derived parameters with model peptides, such as Ac-R-W-V-W-V-N-G-Orn-K(Me)(n)-I-L-Q-NH(2), where n = 0, 1, 2, or 3, were conducted in explicit solvent. Distances between the centers of the indole rings of the two-tryptophan residues, 2 and 4, and the ε-methylated ammonium group on Lys-9 as well as the distance between the N- and C-termini were monitored to estimate the strength and orientation of the cation-π and aromatic/aromatic interactions. In agreement with the experimental data, the stability of the β-hairpin increased significantly with lysine ε-methylation. The ability of MD simulations to reproduce the observed NOEs for the four peptides was further estimated for the monopole-based force fields, AMBER, CHARMM, and OPLSAA. AMOEBA correctly predicted over 80% of the observed NOEs for all 4 peptides, while the three-monopole force fields were 40-50% predictive in only 2 cases and approximately 10% in the other 10 examples. Preliminary analysis suggests that the decreased cost of desolvation of the substituted ammonium group significantly compensated for the reduced cation-π interaction resulting from the increased separation due to steric bulk of the ε-methylated amines.

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Synthetic RNA recognition motifs that selectively recognize HIV-1 trans-activation response element hairpin RNA.

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Synthetic RNA recognition motifs that selectively recognize HIV-1 trans-activation response element hairpin RNA.

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