Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1–3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.
Riboswitches is RNA structural elements generally located in the 5ˈuntranslated region (3ˈUTR) of mRNA. In the genetic regulation, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis for this mechanism requires the ability to study structural changes over time. We apply femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction prior to diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction involving two apo, one ligand-bound intermediate, and the final bound states. These structures support a reaction mechanism model with at least four states and illustrate the structural basis for signal transmission. The two apo conformers differ significantly in the three-way junction and the P1 switch helix relative to the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With a >10-minute delay, the RNA molecules were fully converted to the bound state, in which the substantial conformational changes resulted in conversion of the space group. Such drastic changes in crystallo highlight the important opportunities that micro/nanocrystals may offer in these and similar time-resolved diffraction studies. These results all together demonstrate the potential of "mix-and-inject" time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those involving large conformational changes.
Fifteen years ago, surface acoustic waves (SAW) were found to be able to drive fluids and numerous applications in microfluidics have been developed since. Here, we review the progress made and report on new approaches in setting-up microfluidic, continuous flow acoustic mixing. In a microchannel, chaotic advection is achieved by generation of a SAW driven fluid jet perpendicular to the mean flow direction. Using a high speed video camera and particle image velocimetry, we measure the flow velocities and show that mixing is achieved in a particularly controllable and fast way. The mixing quality is determined as a function of system parameters: SAW power, volume flux and fluid viscosity. Exploring the parameter space of mixing provides a practical guide for acoustic mixing in microchannels and allows for adopting conditions to different solvents, as e.g., required for the generation of nanoscale particles from alcoholic phases. We exemplarily demonstrate the potential of SAW based continuous flow mixing for the production of therapeutic nucleic acid nanoparticles assembled from polymer and lipid solutions.
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