Reusable Microfluidic Chambers for Single-Molecule Microscopy.

Maintaining a consistent environment in single-molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in nonspecific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments, as biotin can be readily incorporated into these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times by using photocleavable biotin (PC-biotin) and UV-light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5 min UV exposure of a specific wavelength. Nonoptimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality as assessed by the nonspecific binding of fluorescently labeled DNA and protein samples and the recovery of the DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging between the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest to the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared to POT1 alone by bridging single- and double-stranded telomeric tracts.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Impact of Divalent Cations on In-Layer Positional Order of DNA-Based Liquid Crystals: Implications for DNA Condensation.

The layered liquid crystalline phases formed by DNA molecules, which include rigid and flexible segments ("gapped DNA"), enable the study of both end-to-end stacking and side-to-side (helix-to-helix) lateral interactions, forming a model system to study such interactions at physiologically relevant DNA and ion concentrations. The observed layer structure exhibits long-range interlayer and in-layer positional correlations. In particular, the in-layer order has implications for DNA condensation, as it reflects whether these normally repulsive interactions become attractive under certain ionic conditions. Using synchrotron small-angle X-ray scattering measurements, we investigate the impact of divalent Mg2+ cations (in addition to a constant 150 mM Na+) on the stability of the inter- and in-layer DNA ordering as a function of temperature between 5 and 65 °C. DNA constructs with different terminal base pairings were created to mediate the strength of the attractive end-to-end stacking interactions between the blunt ends of the gapped DNA constructs. We demonstrate that the stabilities at a fixed DNA concentration of both interlayer and in-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stabilities are even higher at 30 mM Mg2+; however, a marked decrease is observed at 100 mM Mg2+, suggesting a change in the nature of side-by-side interactions within this Mg2+ concentration range. We discuss the implications of these results in terms of counterion-mediated DNA-DNA attraction and DNA condensation.

Structure, Topology, and Stability of Multiple G-quadruplexes in Long Telomeric Overhangs.

Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.

Detecting secondary structure formation with FRET-PAINT.

We present single molecule studies demonstrating the capabilities of the FRET-PAINT method to detect secondary structures that would be challenging to detect with alternative methods, particularly single molecule FRET (smFRET). Instead of relying on the change in end-to-end separation as in smFRET, we use the change in accessibility to a small probe as the criterion for secondary structure formation and relative stability. As a model system, we study G-triplex formation by human telomeric repeat sequences in different structural contexts.

Stability of End-to-End Base Stacking Interactions in Highly Concentrated DNA Solutions.

Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA-DNA interactions at physiologically relevant DNA concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked as well as in-plane ordered duplexes exhibit distinct signatures when studied via small-angle X-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5-65 °C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions, while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 °C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single-molecule studies, which might reflect the elevated DNA concentrations in our studies.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.