RESUMO
Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.
Assuntos
Aptâmeros de Nucleotídeos/química , Compostos Azo/química , Conformação de Ácido Nucleico/efeitos da radiação , RNA/química , Aptâmeros de Nucleotídeos/efeitos da radiação , Fenômenos Biofísicos , Ligantes , Luz , RNA/efeitos da radiação , Riboswitch/efeitos da radiação , Bibliotecas de Moléculas Pequenas/químicaRESUMO
By combining a riboswitch with a cell-permeable photocaged small-molecule ligand, an optochemical gene control element was constructed that enabled spatial and temporal control of gene expression in bacterial cells. The simplicity of this strategy, coupled with the ability to create synthetic riboswitches with tailored ligand specificities and output in a variety of microorganisms, plants, and fungi might afford a general strategy to photocontrol gene expression in vivo. The ability to activate riboswitches by using light enables the interrogation and manipulation of a wide range of biological processes with high precision, and will have broad utility in the regulation of artificial genetic circuits.
Assuntos
Espaço Intracelular/genética , Espaço Intracelular/efeitos da radiação , Riboswitch/efeitos dos fármacos , Riboswitch/efeitos da radiação , Teofilina/farmacologia , Raios Ultravioleta , Permeabilidade da Membrana Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Espaço Intracelular/efeitos dos fármacos , Ligantes , Estrutura Molecular , Riboswitch/genética , Relação Estrutura-Atividade , Teofilina/químicaRESUMO
We report what is to our knowledge the first natural RNA that regulates gene expression in response to intracellular ATP. Using a biochemical screen, we found that several putative riboswitches bind ATP in vitro. The ydaO motif specifically bound ATP and regulated expression of endogenous and reporter genes in response to ATP concentrations in Bacillus subtilis. This discovery demonstrates a role for RNAs in regulating gene expression in response to energy balance in bacteria.
Assuntos
Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos/genética , Bacillus subtilis/genética , RNA Bacteriano/genética , Riboswitch/genética , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Diálise , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Homeostase , Mutação/fisiologia , Conformação de Ácido Nucleico , RNA Bacteriano/química , Riboswitch/efeitos da radiação , Raios UltravioletaRESUMO
Photolabile nucleotides that disrupt nucleic acid structure are useful mechanistic probes and can be used as tools for regulating biochemical processes. Previous probes can be limited by the need to incorporate multiple modified nucleotides into oligonucleotides and in kinetic studies by the rate-limiting step in the conversion to the native nucleotide. Photolysis of aryl sulfide 1 produces high yields of 5-methyluridine, and product formation is complete in less than a microsecond. Aryl sulfide 1 prevents RNA hairpin formation and complete folding of the preQ(1) class I riboswitch. Proper folding is achieved in each instance upon photolysis at 350 nm. Aryl sulfide 1 is a novel tool for modulating RNA structure, and formation of 5-methyluridine within a radical cage suggests that it will be useful in kinetic studies.