RESUMO
Corn, a 28-nucleotide RNA, increases yellow fluorescence of its cognate ligand 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) by >400-fold. Corn was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, in which six unpaired adenosines break overall two-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its interprotomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could provide a basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful for analyzing RNA co-expression or association, or for designing self-assembling RNA nanostructures.
Assuntos
Aptâmeros de Nucleotídeos/química , Dimerização , Corantes Fluorescentes/metabolismo , Quadruplex G , Proteínas Luminescentes/química , Conformação de Ácido Nucleico , Imagem Óptica/métodos , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , Humanos , Proteínas Luminescentes/metabolismo , Proteína Vermelha FluorescenteRESUMO
RNA molecules are essential for cellular information transfer and gene regulation, and RNAs have been implicated in many human diseases. Messenger and non-coding RNAs contain highly structured elements, and evidence suggests that many of these structures are important for function. Targeting these RNAs with small molecules offers opportunities to therapeutically modulate numerous cellular processes, including those linked to 'undruggable' protein targets. Despite this promise, there is currently only a single class of human-designed small molecules that target RNA used clinically - the linezolid antibiotics. However, a growing number of small-molecule RNA ligands are being identified, leading to burgeoning interest in the field. Here, we discuss principles for discovering small-molecule drugs that target RNA and argue that the overarching challenge is to identify appropriate target structures - namely, in disease-causing RNAs that have high information content and, consequently, appropriate ligand-binding pockets. If focus is placed on such druggable binding sites in RNA, extensive knowledge of the typical physicochemical properties of drug-like small molecules could then enable small-molecule drug discovery for RNA targets to become (only) roughly as difficult as for protein targets.
Assuntos
RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Descoberta de Drogas/métodos , Humanos , Ligantes , Proteínas/metabolismoRESUMO
Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second-messenger binding and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule "fragments" that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.
Assuntos
Cristalografia por Raios X/métodos , Descoberta de Drogas/métodos , Riboswitch , Bibliotecas de Moléculas Pequenas/química , Tiamina Pirofosfato/metabolismo , Sítios de Ligação , Ligantes , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Thiamine pyrophosphate (TPP) riboswitches regulate essential genes in bacteria by changing conformation upon binding intracellular TPP. Previous studies using fragment-based approaches identified small molecule "fragments" that bind this gene-regulatory mRNA domain. Crystallographic studies now show that, despite having micromolar Kds, four different fragments bind the TPP riboswitch site-specifically, occupying the pocket that recognizes the aminopyrimidine of TPP. Unexpectedly, the unoccupied site that would recognize the pyrophosphate of TPP rearranges into a structure distinct from that of the cognate complex. This idiosyncratic fragment-induced conformation, also characterized by small-angle X-ray scattering and chemical probing, represents a possible mechanism for adventitious ligand discrimination by the riboswitch, and suggests that off-pathway conformations of RNAs can be targeted for drug development. Our structures, together with previous screening studies, demonstrate the feasibility of fragment-based drug discovery against RNA targets.
Assuntos
Descoberta de Drogas , RNA/antagonistas & inibidores , RNA/metabolismo , Riboswitch , Tiamina Pirofosfato/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Estrutura Molecular , RNA/genética , Reprodutibilidade dos Testes , Especificidade por Substrato , Tiamina Pirofosfato/antagonistas & inibidores , Tiamina Pirofosfato/químicaRESUMO
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.