RESUMO
To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.
Assuntos
DNA Viral/genética , Compostos de Amônio Quaternário/química , Transcrição Gênica , Bacteriófago T4/genética , Benzoxazóis/química , DNA Viral/química , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Compostos de Quinolínio/química , RNA Mensageiro/química , RNA Mensageiro/genética , Raios UltravioletaRESUMO
The release of reactive oxygen species (ROS) or reactive nitrogen species (RNS), i.e., the initial phase of oxidative stress, by macrophage cells has been studied by electrochemistry within a microfluidic device. Macrophages were first cultured into a detection chamber containing the three electrodes system and were subsequently stimulated by the microinjection of a calcium ionophore (A23187). Their production of ROS and RNS was then measured by amperometry at the surface of a platinized microelectrode. The fabricated microfluidic device provides an accurate measurement of oxidative release kinetics with an excellent reproducibility. We believe that such a method is simple and versatile for a number of advanced applications based on the detection of biological processes of secretion by a few or even a single living cell.
