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.

Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding.

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Single-molecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg2+). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1-10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.

Direct unfolding of RuvA-HJ complex at the single-molecule level.

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ∼2-fold. Compared with the ΔGunfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.

MASH-FRET: A Simplified Approach for Single-Molecule Multiplexing Using FRET.

Multiplexed detection has been a big motivation in biomarker analysis as it not only saves cost and labor but also improves the reliability of diagnosis. Among the many approaches for multiplexed detection, fluorescence resonance energy transfer (FRET)-based multiplexing is gaining popularity particularly due to its low background and quantitative nature. Although several FRET-based approaches have been developed for multiplexing, they require either multiple FRET pairs in combination with multiple excitation sources or complicated algorithms to accurately assign signals for individual FRET pairs. Therefore, the need for multiple FRET pairs and multiple excitation sources not only complicates the experimental design but also increases the cost and labor. In this regard, multiplexed sensing by tuning the interdye distance of a single FRET pair could be an ideal solution if identification of multiple FRET efficiencies in a single imaging is possible. Here, implementing a program called MASH-FRET, we evaluated the rigor and capability of this program in identifying seemingly overlapped FRET populations obtained from a multiplexed detection experiment using a single FRET pair. Through MASH-FRET-enabled bootstrap-based analysis of FRET data (also called BOBA-FRET), we demonstrated that the resolution and statistical confidence of the poorly resolved or even unresolved FRET populations can be readily determined. Using simulated FRET data, we further demonstrated that the program can easily identify FRET populations separated by ∼0.1 in mean FRET values, indicating an upper limit of ∼9-fold multiplexing without the need for complicated labeling schemes and multiexcitation sources. Therefore, this paper presents a data analysis approach on an existing platform that has a great potential to simplify the technological needs for multiplexing and to broaden the scope of FRET-based single-molecule analyses.

Single-molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles.

The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif-forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif-forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.

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.

Single-Step FRET-Based Detection of Femtomoles DNA.

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

Homologous Recombination under the Single-Molecule Fluorescence Microscope.

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein-protein and protein-DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein-protein and protein-DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination.

Single-Molecule Imaging Reveals Conformational Manipulation of Holliday Junction DNA by the Junction Processing Protein RuvA.

Interactions between DNA and motor proteins regulate nearly all biological functions of DNA such as gene expression, DNA replication and repair, and transcription. During the late stages of homologous recombination (HR), the Escherichia coli recombination machinery, RuvABC, resolves the four-way DNA motifs called Holliday junctions (HJs) that are formed during exchange of nucleotide sequences between two homologous duplex DNA. Although the formation of the RuvA-HJ complex is known to be the first critical step in the RuvABC pathway, the mechanism for the binding interaction between RuvA and HJ has remained elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) and ensemble analyses, we show that RuvA stably binds to the HJ, halting its conformational dynamics. Our FRET experiments in different ionic environments created by Mg2+ and Na+ ions suggest that RuvA binds to the HJ via electrostatic interaction. Further, while recent studies have indicated that the HR process can be modulated for therapeutic applications by selective targeting of the HJ by chemotherapeutic drugs, we investigated the effect of drug-modified HJ on binding. Using cisplatin as a proof-of-concept drug, we show that RuvA binds to the cisplatin-modified HJ as efficiently as to the unmodified HJ, demonstrating that RuvA accommodates for the cisplatin-introduced charges and/or topological changes on the HJ.

Nascent RNA transcripts facilitate the formation of G-quadruplexes.

Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls.

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions.

Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5'-(TTAGGG)4TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/RNA polymerases with stall forces often <20 pN.

Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update.

Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy.

Single-Molecule FRET-Based Multiplexed Detection.

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field of biosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis. Here, we describe a single-molecule FRET method that enables multiplexed analysis while still using only one FRET pair, and thus the described approach is a significant step forward from conventional FRET methods.

Aptabinding of tumor necrosis factor-α (TNFα) inhibits its proinflammatory effects and alleviates islet inflammation.

Pancreatic islet cell transplantation (ICT) has emerged as an effective therapy for diabetic patients lacking endogenous insulin production. However, the islet graft function is compromised by a nonspecific inflammatory and thrombotic reaction known as the instant blood-meditated inflammatory reaction (IBMIR). Here, we report the characterization of four single-stranded DNA aptamers that bind specifically to TNFα - a pivotal cytokine that causes proinflammatory signaling during the IBMIR process - using single molecule binding analysis and functional assays as a means to assess the aptamers' ability to block TNFα activity and inhibiting the downstream proinflammatory gene expression in the islets. Our single-molecule fluorescence analyses of mono- and multivalent aptamers showed that they were able to bind effectively to TNFα with monoApt2 exhibiting the strongest binding (Kd  âˆ¼ 0.02 ± 0.01 nM), which is ∼3 orders of magnitude smaller than the Kd of the other aptamers. Furthermore, the in vitro cell viability analysis demonstrated an optimal and safe dosage of 100 µM for monoApt2 compared to 50 µM for monoApt1 and significant protection from proinflammatory cytokine-mediated cell death. More interestingly, monoApt2 reversed the upregulation of IBMIR mediating genes induced by TNFα in the human islets, and this was comparable to established TNFα antagonists. Both monoaptamers showed high specificity and selectivity for TNFα. Collectively, these findings suggest the potential use of aptamers as anti-inflammatory and localized immune-modulating agents for cellular transplant therapy.

G-Quadruplex Structure in the ATP-Binding DNA Aptamer Strongly Modulates Ligand Binding Activity.

Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040-0.042 µM for variants as compared to 0.15 µM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies.

Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers.

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis.

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery. This study showcases a computational SELEX method in combination with single-molecule binding studies deciphering effective aptamers against S. aureus IsdA, protein. The established approach demonstrates the ability to expedite aptamer discovery that has the potential to cut costs and predict binding efficacy. The application can be extended to designing aptamers for various protein targets, enhancing molecular recognition, and facilitating the development of high-affinity aptamers for multiple uses.

G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA.

G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.