High FRET State Research Articles

Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes.

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers.

Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteria─Staphylococcus aureus (S. aureus)─which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10-12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Single-Molecule FRET-Based Dynamic DNA Sensor.

Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of diagnostic methods capable of detecting diseases at the molecular level in biological fluids is vital to the emerging revolution in the early diagnosis of diseases. However, the vast majority of the currently available ultrasensitive detection strategies involve either target/signal amplification or involve complex designs. Here, using a p53 tumor suppressor gene whose mutation has been implicated in more than 50% of human cancers, we show a background-free ultrasensitive detection of this gene on a simple platform. The sensor exhibits a relatively static mid-FRET state in the absence of a target that can be attributed to the time-averaged fluorescence intensity of fast transitions among multiple states, but it undergoes continuous dynamic switching between a low- and a high-FRET state in the presence of a target, allowing a high-confidence detection. In addition to its simple design, the sensor has a detection limit down to low femtomolar (fM) concentration without the need for target amplification. We also show that this sensor is highly effective in discriminating against single-nucleotide polymorphisms (SNPs). Given the generic hybridization-based detection platform, the sensing strategy developed here can be used to detect a wide range of nucleic acid sequences enabling early diagnosis of diseases and screening genetic disorders.

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor.

SummaryCircovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsid's conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS-binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS-binding domain. The lack of positively charged amino acids in the HS-binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.

FRET-Based Aptasensor for the Selective and Sensitive Detection of Lysozyme.

Lysozyme is a conserved antimicrobial enzyme and has been cited for its role in immune modulation. Increase in lysozyme concentration in body fluids is also regarded as an early warning of some diseases such as Alzheimer’s, sarcoidosis, Crohn’s disease, and breast cancer. Therefore, a method for a sensitive and selective detection of lysozyme can benefit many different areas of research. In this regard, several aptamers that are specific to lysozyme have been developed, but there is still a lack of a detection method that is sensitive, specific, and quantitative. In this work, we demonstrated a single-molecule fluorescence resonance energy transfer (smFRET)-based detection of lysozyme using an aptamer sensor (also called aptasensor) in which the binding of lysozyme triggers its conformational switch from a low-FRET to high-FRET state. Using this strategy, we demonstrated that the aptasensor is sensitive down to 2.3 picomoles (30 nM) of lysozyme with a dynamic range extending to ~2 µM and has little to no interference from similar biomolecules. The smFRET approach used here requires a dramatically small amount of aptasensor (~3000-fold less as compared to typical bulk fluorescence methods), and it is cost effective compared to enzymatic and antibody-based approaches. Additionally, the aptasensor can be readily regenerated in situ via a process called toehold mediated strand displacement (TMSD). The FRET-based aptasensing of lysozyme that we developed here could be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Single-Molecule Conformational Dynamics of E. coli DNA Polymerase I

The extraordinary accuracy of DNA replication is due in part to the coordinated action of various enzymatic activities exhibited by many DNA polymerases. Structural studies have revealed that many of these functions are localized in spatially separated protein domains, but what is not known is how these domains are coordinated with respect to each other and the DNA substrate in order to ensure efficient and accurate DNA synthesis. One such polymerase is DNA polymerase I (Pol I) from E. coli, which is composed of the Klenow fragment (KF), containing the 5’-3’ polymerase and 3’-5’ exonuclease activities, and a separate 5’ nuclease domain attached via a flexible linker. Here we have used single-molecule FRET (smFRET) microscopy to determine the position of the 5’ nuclease domain of Pol I relative to the downstream strand of various immobilized model DNA substrates. A high-FRET state was observed for a substrate containing a downstream flap - the natural substrate for the 5’ nuclease activity - suggesting that the 5’ nuclease domain is docked with the downstream strand of the duplex, as expected. Interestingly, a similar high-FRET state was also observed for a gapped substrate, suggesting that the 5’ nuclease is also near the downstream strand during polymerase activity. Together, these observations may explain the increased processivity of Pol I versus KF during DNA synthesis and suggest that the 5’ nuclease domain may also serve to mediate the switch between strand-displacement synthesis and RNA primer removal during Okazaki fragment processing. Conversely, no FRET was observed for a substrate containing a double mismatch at the 3’ end of the primer, indicating that the 5’ nuclease domain is remote from the downstream strand of the substrate during proofreading activity.

U2 snRNA Conformation is Regulated by Cus2 to Facilitate Dead-Box Protein Loading

The spliceosome undergoes dynamic changes in composition and conformation during assembly and splicing. We are interested in studying how the spliceosomal U2 snRNP loads onto pre-mRNA branchsites during assembly. This involves multiple conformations of the U2 snRNA (stem IIa vs. stem IIc), the Prp5 DEAD-box protein, and the Cus2 RRM protein. Our results show that Cus2 binds to WT or stem IIa stabilized model RNAs but not to stem IIc. In contrast, Prp5 binds indiscriminately to all three types of U2 RNA and each equally stimulates Prp5's ATPase. Prp5 appears to either displace or out compete Cus2 for U2 RNA binding in an ATP-independent process. Using single molecule FRET, we show the U2 core RNA is dynamic with interconverting populations of high- (likely stem IIc) and mid-FRET states (likely stem IIa). Addition of Cus2 depletes the high-FRET state, consistent with conformational selection of U2 stem IIa by Cus2. Together, our data support spliceosomal proteins acting in concert to facilitate loading of the DEAD-box onto a particular RNA structure. This represents a new paradigm for protein:RNA interactions that is distinct from ordered binding of structure specific RNA-binding proteins (i.e., ribosome assembly) or from positional control of DEAD-box protein loading during exon junction complex formation.

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.

EF-G:A-Site Trna Distance Changes during Translocation

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Attaching a fluorescent probe within domain IV of EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. Two different sites on L11, position 38 or position 87, as well as two different A-site tRNAs, Phe-tRNAPhe or Val-tRNAVal were labeled with Cy5. The EF-G:L11 FRET efficiency is constant during EF-G occupancy of the ribosome (300 - 400 ms), whereas the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. For both tRNAs, the EF-G:tRNA pair spends most of its time in the intermediate FRET state, with shorter lifetimes in the high and low FRET states. Each transition in the FRET value corresponds to a relative distance change of about 5 to 7 A, so in total, we capture a 12 A relative movement of A-site tRNA away from EF-G during translocation. EF-G:A-site tRNA FRET is sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state. In addition, the translocation inhibitors viomycin and spectinomycin block formation of the low FRET state, although spectinomycin allows more movement between EF-G and tRNA than viomycin. Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

Dynamics of the N-Terminal Domain of Myosin V Monitored by FRET

The N-terminus is a highly variable region among the myosin superfamily and has been associated with alterations in rotation of the lever arm and step size. We examined the structural dynamics of the N-terminal region of myosin V using steady-state and stopped-flow FRET. We introduced a tetra-cysteine site at the extreme N-terminus of a Myosin V 1IQ construct and labeled it with a bisarsenical fluorescin derivative (FlAsH) (MV NT-FLAsH). Energy transfer between FlAsH and mant or deac labeled nucleotides (dmantATP, dmantADP and deacATP) was monitored. Steady-state FRET experiments with the mant-FlAsH pair demonstrated a high FRET state in the presence of dmantATP and a low FRET state in the presence of dmantADP. Sequential-mix, in which MV NT-FLAsH is first mixed with dmantATP, aged to allow formation of the M.ADP.Pi state, and then mixed with saturating actin allowed us to explore the actin-acitvated product release steps. Biphasic transients with rates corresponding to the fast and slow rate of dmantADP release were observed. The sequential mix experiments with deacATP yielded three phases; a fast, actin dependent, phosphate release phase followed by the two phases of ADP release. Our results suggest the FRET signal monitors a structural change in the N-terminus associated with the sequential release of products. A high FRET state upon binding of ATP is associated with formation of the pre-powerstroke state of the lever arm and a low or no FRET state in the presence of ADP suggests formation of the post-powerstroke state. Our results allow us to hypothesize that movement of the N-terminal domain follows the movement of the converter/lever arm. Further experiments will explore the dynamics of the N-terminal domain and examine how its motion correlates with the conformation of the nucleotide binding pocket and lever arm.

Kinetics of the Triplex-Duplex Transition in DNA

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.

Freeing the Promoter Site: Mechanistic Insights into the Interaction of Mot1 with TBP using spFRET

Gene transcription is a central process of life and is highly regulated with a multitude of transcription factors. Recently, we could show that dynamics of the Tata-box Binding Protein (TBP) on the promoter site also plays a role in gene regulation. Using single-pair Forster Resonance Energy Transfer (spFRET) experiments, we have monitored the conformation of TBP bound to the H2B promoter site in the presence and absence of Mot1. Upon the binding of Mot1 to the TBP-DNA complex, a high FRET state between TBP and labeled DNA is formed. Contrary to what was expected, Mot1 bound to TBP-DNA complexes was unable to dissociate TBP from the DNA upon addition of ATP. Instead, the TBP-DNA complex returned to its original conformation even though Mot1 remain bound to the complex. Only when Mot1 was also present at nM concentrations in solution was dissociation of TBP from the promoter site observed upon addition of ATP. This suggests that a single Mot1 is either insufficient or inefficient at dissociating TBP alone.We also used spFRET to monitor the DNA conformation during the interaction of Mot1 with TBP-DNA. TBP-bound DNA was observed to fluctuate between three conformations. The FRET values of the conformation varied depending on the orientation of TBP on the promoter. Fluctuations between the same conformations were observed when Mot1 was bound to the TBP-DNA complex but with an altered kinetics. The results suggest that Mot1 primes incorrectly bound TBP for dissociation, preferably freeing promoter sites with an inappropriately bound TBP molecule.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

DNA Methylation Induces a More Compact and Rigid Nucleosome Structure

Cytosine methylation in CpG dinucleotides is an important epigenetic modification in eukaryotes with roles in regulating a variety of genome transactions. There have been many studies of DNA methyltransferases and methyl-CpG binding proteins elucidating their roles in various genome activities. However, less is known about how methylated CpGs directly affect nucleosome structure. We implemented a single molecule FRET coupled with anisotropy that can simultaneously measure dynamic distance changes and flexibility of the two ends of a nucleosomal DNA. Using the method, we monitored effects of DNA methylation on the structure of mononucleosomes. In the absence of methylation, most nucleosomes displayed two low FRET states (FRET efficiency <0.5). When treated with a CpG methyltansferase, we observed a >40-fold increase in the number of nucleosomes that made excursions to a high FRET state (FRET efficiency >0.7). Moreover, based on the anisotropy measurements, a stronger association between the DNA ends and the histone octamer was observed from the nucleosomes in the high FRET state. The increased FRET and anisotropy after DNA methylation strongly suggest a more compact and rigid nucleosomal structure and provide a basic biophysical understanding of how DNA methylation may contribute to the formation of a repressive and transcriptionally inactive chromatin structure.

Temperature Dependent Energy Transfer Measurements Reveal Flexibility in the Upper 50 kDa Domain of Myosin V

Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides and IAEDANS actin. These FRET studies suggest that myosin V can adopt a conformation in which the nucleotide binding pocket and the actin binding cleft are in a closed conformation. Our studies suggest the upper 50 kDa domain may be highly flexible in certain nucleotide-states which allows tight binding to nucleotide and actin. Molecular geometric simulations demonstrate the upper 50 kDa domain is most flexible in the myosin V.ADP state, consistent with this state having a high affinity for ADP and actin. Currently, we examined the temperature dependence of the FRET signal between mantADP and MV FlAsH. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-37°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. We also found that FlAsH labeled G440A MV, a non-hydrolyzable mutant, has a similar temperature-dependent transition in the presence of mantATP. In contrast, the transition does not occur in the presence of mantADP.BeFx or with the non-hydrolyzable E44A MV mutant in the presence of mantATP. Our results suggest coordination of the gamma-phosphate of ATP rigidifies the upper 50 kDa domain which results in a weak actin affinity state (open actin binding cleft and closed nucleotide binding pocket). However, upon phosphate release the upper 50kDa domain becomes more flexible which allows myosin to adopt a conformation in which it has a high affinity for both nucleotide and actin (closed nucleotide binding pocket and actin binding cleft).

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time.

Many questions in molecular and cellular biology can be reduced to questions about 'who talks to whom, when and how frequently'. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA-protein interactions in real time.