Single-molecule FRET has been widely used for monitoring proteinnucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0-3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG-I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.cis-trans isomerization | DNA-protein interaction | RNA-protein interaction | label free protein S ingle-molecule Förster resonance energy transfer (FRET) has been a powerful tool in probing protein-nucleic acid interactions as demonstrated by numerous studies revealing unexpected dynamic movement and conformational changes of proteins that cannot be resolved by conventional ensemble techniques (1-4). Despite such advantages, FRET measurement is often limited to proteins that can be site-specifically labeled and the protein's interaction with the corresponding DNA or RNA in a FRETsensitive distance range of 3 to 8 nm (5). The conjugation of fluorescent dye to a protein involves mutagenesis (6) and/or chemical modifications (7), which may disrupt the structure and function of the protein. Furthermore, the labeling procedures are not straightforward and are often labor intensive yet with a low yield.Recently we developed an alternative single molecule assay termed protein induced fluorescence enhancement (PIFE) whereby the emission of a fluorescent dye reports on its proximity to an interacting protein; i.e., the dye becomes brighter when a protein approaches its vicinity (8). This photophysical effect was originally employed in stop flow measurement (9) for monitoring directional movement of DNA motor proteins in ensemble (10-13), for following the dynamics of DNA and RNA polymerases on DNA (14, 15), and for detecting the motion of helicases on RNA or DNA at the single-molecule level (16,17). This photophysical characteristic is exhibited by fluorophores such as Cy3, which undergoes cis-trans isomerization reaction. External factors such as protein reduce the rate at which the fluorophore isomerizes from the photo-acti...

We propose to utilize azobenzene as a nanomolecular switch which can be triggered by transmitting electrons above threshold biases. The effect is explained by an electron impact trans-cis conformational change of the isolated azobenzene molecules. The molecular electronic states of both isomers have been measured with spatially resolved scanning tunneling microscopy or spectroscopy, leading to suggested transition pathways of the electron-induced isomerization.

Sequence recognition through base pairing is essential for DNA repair and gene regulation but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized despite its potential importance in nucleic acids-based biotechnologies and gene silencing. Here we use single molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to quantify precisely the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement of seven nucleotides complementarity to seed gene silencing by small non-coding RNA and may help guide performance improvement in DNA and RNA-based bio- and nano-technologies where off-target effects can be detrimental.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes.

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Carbon nanodots (CDs) were initially synthesized by dehydrating carbohydrates using a commercial household microwave (700 W). To prepare BN-CD, 960 mg of citric acid (5.0 mmol, Aldrich) and 310 mg of boric acid (5.0 mmol) were dissolved in 10 mL of water. To this transparent solution, 347 µL of EDA (5.0 mmol) was added under vigorous stirring for 2 min. The solution was placed into a microwave oven and heated for 2 min, and a yellow solid was obtained after cooling to room temperature. The solid was diluted in 5.0 mL of water. The yellow suspension was dialyzed (SpectraPore MWCO 500 -1,000) for 2 days to remove salts and unreacted chemicals. To synthesize N-CD, microwave pyrolysis was performed in the absence of boric acid. BN-CD0.5 and BN-CD2 were prepared with 2.5 mmol (0.5 equiv. of citric acid and ethylene diamine) and 10 mmol (2 equiv. of citric acid and ethylene diamine) of boric acid, with the same concentrations of other precursors as described above. Non-doped plain CD was synthesized with 5 mmol of citric acid through hydrothermal method at 180 o C for 6 hr. B-CD was synthesized with 5 mmol of boric acid and citric acid.

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.