Affinity chromatography in dynamic combinatorial libraries: one-pot amplification and isolation of a strongly binding receptor

We report the one-pot amplification and isolation of a nanomolar receptor in a multibuilding block aqueous dynamic combinatorial library using a polymer-bound template. By appropriate choice of a poly(N,N-dimethylacrylamide)-based support, unselective ion-exchange type behaviour between the oppositely charged cationic guest and polyanionic hosts was overcome, such that the selective molecular recognition arising in aqueous solution reactions is manifest also in the analogous templated solid phase DCL syntheses. The ability of a polymer bound template to identify and isolate a synthetic receptor via dynamic combinatorial chemistry was not compromised by the large size of the library, consisting of well over 140 theoretical members, demonstrating the practical advantages of a polymer-supported DCL methodology

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.This work was supported by grants from Science Foundation Ireland [12/IP/1492 and 13/1A/1853 to J.F.A; 12/IA/1335 to P.V.B.], US. National Institutes of Health [RO3 MH098688 to J.F.A.], the Wellcome Trust [106207 to A.E.F and 094423 to P.V.B.] and the European Research Council (ERC) grant No. 646891 to A.E.F.]This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/nar/gkw53

Small molecule evolution: dynamic combinatorial selection applied to the discovery of novel RNA-binding small molecules.

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.RNA is no longer simply an intermediary between the genetic code of DNA and proteins; we are living in an RNA world. Ribonucleic acids have been shown to be involved in many important cellular processes such as transcriptional and translational regulation, enzymatic catalysis, protein function, and various essential interactions in cellular machinery and disease states. Selective and high affinity small molecule ligands for RNA binding have utility as tools in the understanding of fundamental RNA interactions, and in the development of novel therapeutics. As such, the development of small molecules capable of binding RNA sequences with high affinity and selectivity is currently a large and growing area of bioorganic chemical research. Dynamic combinatorial chemistry (DCC) is an emerging technique that allows both the synthesis and screening of small molecule libraries in situ. Governed by thermodynamic control, dynamic combinatorial chemistry employs reversible assembly of starting materials into a dynamic combinatorial library (DCL). Addition of a target biomolecule to the DCL enhances production of library members with affinity for the target; in essence it drives small molecule evolution. The research described herein developed a novel Resin Bound Dynamic Combinatorial Chemistry (RBDCC) strategy for the development of small molecule ligands for two biomedically important RNA hairpin sequences: a frameshift inducing stemloop involved in HIV-1 translation, and a (CUG)n triplet repeat RNA involved in myotonic dystrophy pathogenesis. An 11,325 member disulfide exchange based Resin Bound Dynamic Combinatorial Library (RBDCL), the largest reported to date, was synthesized and screened against these two important disease related RNA hairpin structures. In the context of HIV, screening the 11,325 member RBDCL selected a small molecule ligand that binds the target HIV-1 frameshift inducing RNA stemloop with high affinity (low μM dissociation constants), and significant selectivity over other related RNA hairpins. Additionally, in the context of myotonic dystrophy, library screening selected a series of ligands that bind the target (CUG)n RNA with high affinity (low μM dissociation constants), and good selectivity over other RNA sequences. Importantly, the (CUG)n ligands were shown to inhibit the interaction of (CUG)n RNA and the splicing factor MBNL1 in vitro. The (CUG)n – MBNL1 interaction causes misregulated splicing in myotonic dystrophy, and as such, compounds that inhibit the interaction hold promising therapeutic utility. Together, this research highlights the utility of the RBDCC library synthesis and screening approach to develop novel small molecule ligands with high affinity and selectivity for two biomedically important RNA sequences

Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRNAs. Here, we report the use of a small molecule microarray (SMM) screening approach to identify low molecular weight compounds that directly bind to a pre-miR-21 hairpin. Compounds identified using this approach exhibit good affinity for the RNA (ranging from 0.8–2.0 μM) and are not composed of a polycationic scaffold. Several of the highest affinity compounds inhibit Dicer-mediated processing, while in-line probing experiments indicate that the compounds bind to the apical loop of the hairpin, proximal to the Dicer site. This work provides evidence that small molecules can be developed to bind directly to and inhibit miR-21

A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities

Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule–protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD’s “bump” ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck