Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

Release of Highly Active Ice Nucleating Biological Particles Associated with Rain

Biological particles may play an important role in the climate system by efficiently acting as ice nucleating particles (INPs) at a higher temperature range (e.g., above −20 °C where representative INPs such as mineral dust remain inactive), but there is an obvious lack of direct evidence that these particles serve in this manner. Here, we collected ambient particles under different weather conditions for identifying INPs that are active above −22 °C. The abundance of such efficient INPs increased during or following rainfall events. The extensive characterization of individual particles by three different analyses (particle morphology and composition, heat sensitivity of ice nucleation activities, and biological fingerprinting by DNA staining) revealed that efficient INPs have distinctly biological characteristics, which differ significantly from more abundant, representative, and relatively less active INPs, such as mineral dust. Additionally, by combining the heat-sensitivity experiments and DNA staining techniques, efficient INPs were found to contain heat-sensitive biomaterials and biological cells. Our findings provide direct evidence that biological particles are preferentially released into the atmosphere during rainfall events and act as important atmospheric INPs at higher temperature ranges (warmer than −22 °C), where typical INPs remain inactive.