RESUMEN
RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.
Asunto(s)
Azidas/química , Diazometano/análogos & derivados , Etiquetas de Fotoafinidad/química , ARN/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Azidas/metabolismo , Azidas/efectos de la radiación , Sitios de Unión , Diazometano/metabolismo , Diazometano/efectos de la radiación , Ligandos , Simulación del Acoplamiento Molecular , Etiquetas de Fotoafinidad/metabolismo , Etiquetas de Fotoafinidad/efectos de la radiación , Prueba de Estudio Conceptual , ARN/química , Transcripción Reversa , Análisis de Secuencia de ADNRESUMEN
DNA-encoded libraries (DELs) are collections of small molecules covalently attached to amplifiable DNA tags carrying unique information about the structure of each library member. A combinatorial approach is used to construct the libraries with iterative DNA encoding steps, facilitating tracking of the synthetic history of the attached compounds by DNA sequencing. Various screening protocols have been developed which allow protein target binders to be selected out of pools containing up to billions of different small molecules. The versatile methodology has allowed identification of numerous biologically active compounds and is now increasingly being adopted as a tool for lead discovery campaigns and identification of chemical probes. A great focus in recent years has been on developing DNA compatible chemistries that expand the structural diversity of the small molecule library members in DELs. This chapter provides an overview of the challenges and accomplishments in DEL technology, reviewing the technological aspects of producing and screening DELs with a perspective on opportunities, limitations, and future directions.
Asunto(s)
ADN/genética , Descubrimiento de Drogas , ADN/química , Biblioteca de Genes , Técnicas de Síntesis en Fase SólidaRESUMEN
The center of DNA three-way junctions, constituting a yoctoliter (10(-24) L) volume, is applied as an efficient reactor to create DNA-encoded libraries of chemical products. Amino acids and short peptides are linked to oligonucleotides via cleavable and noncleavable linkers. The oligonucleotide sequences contain two universal assembling domains at the center and a distal codon sequence specific for the attached building block. Stepwise self-assembly and chemical reactions of these conjugates in a combinatorial fashion create a library of pentapeptides in DNA three-way junctions in a single reaction vessel. We demonstrate the formation of an evenly distributed library of 100 peptides. Each library member contains a short synthetic peptide attached to a unique genetic code creating the necessary "genotype-phenotype" linkage essential to the process of in vitro molecular evolution. Selective enrichment of the [Leu]-enkephalin peptide from an original frequency of 1 in 10 million in a model library to a final frequency of 1.7% in only two rounds of affinity selection is described and demonstrates successful molecular evolution for a non-natural system.