Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

DNA polymerases at work: single-molecule observations of DNA synthesis in real time

This thesis focuses on the characterization of DNA polymerases with single-molecule techniques. More specifically, I aimed to study polymerase processivity and fidelity-related conformational changes using assays based on Förster Resonance Energy Transfer (FRET) on a total internal reflection fluorescence (TIRF) microscope. Chapter 2 reviews some of the recent applications of single-molecule FRET (smFRET) to study DNA and DNA binding proteins, in particular DNA polymerases. The chapter begins with an introduction of FRET, employed to measure distance changes in the 1-10 nm region, and introduces the two most common fluorescence-based implementations of single-molecule techniques: confocal microscopy and TIRF microscopy. The chapter concludes with a short discussion on FRET-based structural modelling, parts of which are applied in practice later in this thesis. In chapter 3, I report the development of a short, fluorescently labelled DNA sensor to probe DNA polymerization at the single-molecule level. The sensor is a simple primer-template combination labelled with donor and acceptor fluorophores suitable for FRET. The advantage of this assay is that polymerases do not need to be labelled with any fluorophore. I show that the FRET efficiency of the sensors changes significantly upon polymerization of the 25 nucleotide template, and I present time traces showing polymerization of single sensors by three different polymerases (E. coli DNA Polymerase I (KF), human Polymerase Beta (POLB) and the α subunit of bacterial Polymerase III (POLIIIα)). Based on these traces, I can measure polymerase speed and pausing: KF and POLIIIα extended the primer in ~1.0-1.5 s, but POLB was far slower (tens of seconds). I foresee applications for these sensors in the single-molecule field, where they can be used to characterize the processivity of other polymerases, but also for ensemble experiments in which native polymerases need to be tested for activity. I take a closer look at POLB in chapter 4. This polymerase is involved in DNA repair, and I address the question whether resolving the conformational dynamics of the enzyme can shed new light on fidelity-related mechanisms. Previous work on both KF and POLB showed that the polymerase “fingers” domain binds a nucleotide and subsequently transfers it to the active site (a conformational change known as “fingers closing”). For KF, it was shown that the fingers domain does not entirely close when non-complementary nucleotides are present, suggesting that nucleotides are screened for complementarity with the templating base during fingers closing. To see whether POLB employs a similar mechanism, I designed an smFRET assay with an immobile donor fluorophore on the DNA primer and an acceptor fluorophore on the fingers domain. Using this approach, I can visualize fingers closing in the presence of the correct nucleotide in single POLB-DNA complexes. Incorrect nucleotides (non-complementary dGTPs and complementary rUTPs) did not induce fingers closing. Instead, we observed a slight shift in the mean FRET efficiency of the open conformation (from E* ≈ 0.55 to E* ≈ 0.62), while a fully closed conformation corresponds to E* ≈ 0.75. I find evidence for a partially closed, fidelity-related conformation of the fingers subdomain. Simultaneously, I find that high concentrations of incorrect nucleotides (1 mM and 3 mM) stabilize the POLB-DNA complex by lowering the POLB dissociation rate. In contrast, for KF, a destabilizing effect was shown previously. The mechanism behind this stabilization remains unknown, but I hypothesize that with the abundance of incorrect nucleotides in the cell, DNA repair is much faster if high levels of these nucleotides do not promote dissociation. In chapter 5, I introduce novel nanofluidic devices for high-throughput single-molecule imaging. These devices are completely made of glass. I present two designs: one design with a parallel array of nanochannels for equilibrium studies, and another with a single, T-shaped nanochannel for mixing studies allowing access to non-equilibrium conditions. A channel height of 200 nm confines movement of the molecules such that they do not move out of focus. With the implementation of parallel flow control, the devices can be driven with conventional syringe pumps. I achieve a high temporal resolution on our emCCD camera due to stroboscopic excitation (1.5 ms excitation in 10 ms frame time). I show that we can track single molecules at low concentrations for extended periods of time. The track length depends on the flow speed, but ranges from several frames to tens of frames. Moreover, at higher concentrations, I achieve hundreds of thousands of localizations within 10 minutes. These localizations allowed me to construct flow profiles, which confirms that the flow in the nanochannels is laminar. I also calculate that, at low flow rates and with the small DNA molecules I used, motion due to flow is of the same order of magnitude as motion due to diffusion. I illustrate this concept by mixing DNA hairpins in a primarily open configuration with a high-salt solution in the mixing channel: the FRET signature of the hairpins changes abruptly towards an equilibrium of primarily closed DNA hairpins. After fine-tuning the conditions, this so-called “diffusive” mixing is employed to trigger single-molecule reactions: I successfully polymerize my previously described DNA sensor inside the channel. I believe that these nanofluidic devices are a promising platform for studying non-immobilized single molecules at high throughput and high temporal resolution.</p

High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods