Colloidal semiconductor quantum dots are attractive fluorophores for multicolor imaging because of broad absorption and narrow emission spectra, they brighter far more photostable than organic dyes. However, severe intermittence in (also known as blinking) has been universally observed from single considered an intrinsic limitation difficult to overcome. This is unfortunate growing applications spectroscopy biological molecules information processing using photon sources could greatly...

Despite the recent advances in single-molecule manipulation techniques, purely mechanical approaches cannot detect subtle conformational changes biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence that allowed us to examine effect subpiconewton forces on nanometer scale motion Holliday junction (HJ) at 100-hertz bandwidth. The HJ is an exquisitely sensitive sensor whose response amplified with increase its arm lengths, demonstrating...

Six interfluorophore FRET efficiencies Eij (see scheme) can be determined in real time by a single-molecule four-color technique both confocal and total-internal-reflection fluorescence microscopy. This was used to probe the correlated motion of four arms Holliday junction, assess correlation RecA-mediated strand exchange events at ends synaptic complex.

The viral sensor MDA5 distinguishes between cellular and dsRNAs by length-dependent recognition in the range of ∼0.5–7 kb. ability to discriminate dsRNA length at this scale sets apart from other receptors immune system. We have shown previously that forms filaments along disassemble upon ATP hydrolysis. Here, we demonstrate filament formation alone is insufficient explain its specificity, because intrinsic affinity for depends only moderately on length. Instead, uses a combination end...

Abstract MicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at post-transcriptional level. Differential profiles miRNAs across a range diseases have emerged as powerful biomarkers, making reliable yet rapid profiling technique for potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging can profile purified endogenous with high sensitivity, specificity, and reliability. Compared to...

DOT1L is a histone H3 Lys79 methyltransferase whose activity stimulated by H2B Lys120 ubiquitination, suggesting cross-talk between methylation and ubiquitination. Here, we present cryo-EM structures of complexes with unmodified or ubiquitinated nucleosomes, showing that recognizes ubiquitin the H2A/H2B acidic patch through C-terminal hydrophobic helix an arginine anchor in DOT1L, respectively. Furthermore, combined single-molecule FRET experiments show ubiquitination enhances noncatalytic...

Abstract Colloidal semiconductor quantum dots are promising for single‐molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept fluorescence resonance energy transfer (FRET) applications, we measured FRET between single dot organic fluorophore Cy5. DNA Holliday junction dynamics with the dot/Cy5 pair identical those obtained conventional Cy3/Cy5 pair, that is, conformational changes individual molecules can be observed by using as donor.

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing three-color FRET techniques, however, suffer from severe photobleaching Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time improved data quality....

Z-DNA, a left-handed isoform of Watson and Crick’s B-DNA, is rarely formed without the help high salt concentrations or negative supercoiling. However, Z-DNA-binding proteins can efficiently convert specific sequences B conformation into Z in relaxed DNA under physiological conditions. As case many other interactions coupled with structural rearrangements biology, it has been an intriguing question whether actively induce Z-DNAs passively trap transiently preformed Z-DNAs. In this study, we...

Significance RNA molecules fold into functional structures as they are being synthesized by transcription. Here, we developed a single-molecule fluorescence resonance energy transfer (FRET) assay that permits real-time monitoring of the cotranscriptional folding molecules. This FRET makes it possible to carry out controlled studies two main physical mechanisms regulate folding: transcriptional pausing and speed.

Abstract Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism action are unclear. Diverse models have been proposed for the initial interaction between RNA polymerase (RNAP) and (catch-up stand-by pre-terminational models); terminational release transcript (RNA shearing, RNAP hyper-translocation or displacing, allosteric post-terminational outcome (whether dissociates remains bound to DNA). Here, we use single-molecule fluorescence assays study...

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but mechanism by which facilitates sRNA still debated. We developed a fluorescence resonance energy transfer assay to probe how modulates interaction between sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes intra- intermolecular structures can be monitored real time. Our data show promotes strand exchange reaction internal structure rpoS replaced...

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part its essential cellular functions, the enzyme generates DNA breaks, but regulation this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence events during cleavage by topoisomerase II: nonspecific binding, sequence-specific bending, and stochastic We have identified unexpected structural roles Mg 2+...