Ideally, a single-molecule platform would be simple to implement (in terms of hardware, method, and analysis) and mimic cell-like conditions. This inspired us to develop Convex Lens-induced Confinement (CLiC) microscopy, to enable direct visualization, manipulation, and quantification of biomolecules at the single molecule level. CLiC overcomes the limitations of existing techniques like TIRF (total internal reflection fluorescence), confocal microscopy, and optical/magnetic trapping. By mechanically confining molecules to nanoscale wells (and other features), CLiC enables long observation times (from seconds to hours) of untethered and freely diffusing biomolecules. Looking at hundreds of copies of molecules (or more) at once, it provides high statistics and good signal-to-noise, enabling dissection of complex processes. By enabling reagent exchange and control over confinement geometry, CLiC mimics crowded conditions in cells. In this work, we use CLiC imaging to investigate the role of DNA structure in mediating the binding activity of small molecules (oligonucleotide probes, proteins) to target sites on DNA, building on our recent publication (Scott et. al., Nucleic Acids Research, 2018). We study the impact of temperature and DNA supercoiling upon the binding affinity and kinetics of small molecules to individual, untethered DNA plasmids. By interpreting our microscopy results with supporting simulations, we investigate the role of specific higher-order structures (such as ZDNA) in actively mediating site-unwinding; for instance, we establish that ZDNA is prevalent at low temperature, but suppressed at high temperature. Further, we explore how oligonucleotide sequence, solution salinity, crowding agents, and other cofactors (such as oligo-binding proteins) impact the interaction kinetics and affinities. Looking ahead, we extend our CLiC nucleic acid assay to interrogate and quantify the binding of modified DNA to RNA targets, a fundamental parameter in understanding the mechanisms and efficacies of nucleic acid therapeutics. 2350-PosTemperature Dependence of the Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins mNeptune1, mNeptune2.5 and mCardinal2 Chandra Dhakal, Prem Chapagain, Xuewen Wang. Physics, Florida International University, Miami, FL, USA. Low temperature experiments on mneptune1, mNeptune2.5 and mcardinal2 show a reduced stokes shift compared to room temperature. This suggests that the increased flexibility of the chromophore environment at higher temperatures along with its ability to reorganize after excitation is related to the larger stokes shift. We used molecular dynamics (MD) simulations to investigate the dynamics of the hydrogen bonds formed in the chromophore regions of the farred fluorescent proteins mNeptune1, mneptune2.5 and mcardinal2. We explored the protein-chromophore hydrogen bond pattern at various temperatures and correlate the hydrogen bond dynamics to the experimentally observed Stoke's shifts. Spider silks form biomaterials including fibres, fil...
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