Dataset Information

Combinatorial Synthesis, Screening, and Binding Studies of Highly Functionalized Polyamino-amido Oligomers for Binding to Folded RNA.

ABSTRACT: Folded RNA molecules have recently emerged as critical regulatory elements in biological pathways, serving not just as carriers of genetic information but also as key components in enzymatic assemblies. In particular, the transactivation response element (TAR) of the HIV genome regulates transcriptional elongation by interacting specifically with the Tat protein, initiating the recruitment of the elongation complex. Preventing this interaction from occurring in vivo halts HIV replication, thus making RNA-binding molecules an intriguing pharmaceutical target. Using ?-amino acids as starting materials, we have designed and synthesized a new class of polyamino-amido oligomers, called PAAs, specifically for binding to folded RNA structures. The PAA monomers were readily incorporated into a 125-member combinatorial library of PAA trimers. In order to rapidly assess RNA binding, a quantum dot-based fluorescent screen was developed to visualize RNA binding on-resin. The binding affinities of hits were quantified using a terbium footprinting assay, allowing us to identify a ligand (SFF) with low micromolar affinity (k(d) = 14??M) for TAR RNA. The work presented herein represents the development of a flexible scaffold that can be easily synthesized, screened, and subsequently modified to provide ligands specific for binding to folded RNAs.

SUBMITTER: Pokorski JK

PROVIDER: S-EPMC3432390 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Combinatorial Synthesis, Screening, and Binding Studies of Highly Functionalized Polyamino-amido Oligomers for Binding to Folded RNA.

Pokorski Jonathan K JK Appella Daniel H DH

Journal of nucleic acids 20120821

Folded RNA molecules have recently emerged as critical regulatory elements in biological pathways, serving not just as carriers of genetic information but also as key components in enzymatic assemblies. In particular, the transactivation response element (TAR) of the HIV genome regulates transcriptional elongation by interacting specifically with the Tat protein, initiating the recruitment of the elongation complex. Preventing this interaction from occurring in vivo halts HIV replication, thus m ...[more]

PMID: 22957210

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Project description:Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

Dataset Information

Combinatorial Synthesis, Screening, and Binding Studies of Highly Functionalized Polyamino-amido Oligomers for Binding to Folded RNA.

Publications

Combinatorial Synthesis, Screening, and Binding Studies of Highly Functionalized Polyamino-amido Oligomers for Binding to Folded RNA.

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