Soma Dhakal

• Professor (Assistant) at Virginia Commonwealth University

Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this work, we utilized fluorescence spectroscopy to measure the concentration of quinine in commercial t...

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 electrochemic...

Branch migration (BM) of DNA Holliday junctions (HJs) occurs through base-pair rearrangements between homologous DNA molecules during the repair of double-strand breaks (DSBs) by homologous recombination. Despite the fact that BM is conserved among organisms and is essential to stabilize recombination intermediates, which occurs by avoiding the rev...

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 struct...

The repair of double-stranded DNA breaks (DSBs) 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 other...

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 b...

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 molecu...

Fluorescence resonance energy transfer (FRET) has become a powerful tool for visualizing molecular interactions, single-molecule conformational dynamics, binding, and other molecular signaling events. However, until recently, FRET spectroscopy/microscopy has been mainly used for monitoring single events. Recent pursuits on simultaneous detection an...

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)-base...

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...

Multiplex detection of biomolecules is important in bionanotechnology and clinical diagnostics. Multiplexing using engineered solutions such as microarrays, synthetic nanopores, and DNA barcodes are promising but they require sophisticated design/engineering and typically yield semi-quantitative information. Single-molecule FRET (smFRET) is an attr...

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined re...

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor–stator units that consume chemical energ...

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 f...

In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-Assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we...

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a syste...

Supplementary Figures 1-61, Supplementary Tables 1-4, Supplementary Notes 1-4, Supplementary Methods and Supplementary References

The control of enzymatic reactions using nanoscale DNA devices offers a powerful application of DNA nanotechnology uniquely derived from actuation. However, previous characterization of enzymatic reaction rates using bulk biochemical assays reported suboptimal function of DNA devices such as tweezers. To gain mechanistic insight into this deficienc...

DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanopar...

Switchable nanomachines provide a platform to control dynamic functional states by altering distances at the nanoscale on demand. Recently, a tweezers-like DNA device was used to control the activity of an enzyme/cofactor pair juxtaposed on the two arms of the tweezers. Initial studies focused on bulk properties of the tweezers-mediated reactions,...

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 ple...

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 area...

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....

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...

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)(4)TTA, along three different trajectories. After the end-to-end...

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non...

A typical force-extension (F-X) curve obtained from the mechanical unfolding of the secondary structure in the ILPR-I3 (5'-TGT CCCC ACA CCCC TGT CCCC ACA) at pH 5.5. The unfolding event (∼5 nm) is highlighted by a dashed green circle. Black curve is the WLC fitting of the relaxing curve. Inset is the schematic of the laser tweezers experiment. (DOC...

CD spectra of sequences described in Table 1. CD spectra of the mutants with mutation sites in each of the three C4 tracts in the ILPR-I3 are plotted in A), B), and C), respectively. The spectra of ILPR-I3 (red) and the scrambled sequence (black) are also included in each Figure for direct comparison. These CD experiments were performed at 5 µM oli...

Calculation of contour length change (ΔL) of the four possible candidates shown in Figure S4. (DOC)

Four possible structures that employ C:CH+ pair stacking in the ILPR-I3 sequence. The three C4 tracts are shown in blue and other regions are shown in red for structures in (A)–(D). The unfolding direction for each structure is shown by black arrows labeled with “F”. Notice the structures with free C4 tracts at the 5'-end yield ΔL values identical...

Calculation of the unfolding rate constant (kunfold) at 0 pN for the intramolecular i-motif (“ILPR-I4”, calculation based on published data (References S1References S1 11)) and the 45 pN population in the ILPR-I3/ILPR-I1 mixture (“ILPR-I3+ILPR-I1”) from the plot of ln[r ln(1/N)] versus rupture force. We used the equation S2 to estimate the kunfold,...

Electrophoretic mobility shift assays (EMSA) of the ILPR-I3. EMSA of the ILPR-S3 (scrambled DNA, lane 1) and the ILPR-I3 (lane 2) at 1 µM strand concentration. Lane 3 is the DNA marker (M). Left panel, a native gel at pH 5.5. Right panel, a denatured gel (10% PAGE, 7 M urea). (DOC)

Intensity scan for ILPR-I3 bands (the green trace to the left of the gel) and the fold protection for ILPR-I4 (I4, black) and ILPR-I3 (I3, green) for Br2 footprinting in a 10 mM sodium phosphate buffer at pH 5.5 with 100 mM KCl. Note that this gel is identical with that in Figure 5B (see Materials and Methods for fold protection calculation). Sampl...

Supporting Information References. (DOC)

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 i...

Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are...

In 1910 Ivar Bang reported that concentrated solutions of guanylic acid formed a gel. It took half a century before Gellert et al. in 1962 discovered the structural motif, a guanine-quartet, to be the basis for guanylic acid gelation. This chapter starts with a historical overview and gives credit to Ivar Bang and several other pioneers in the fiel...

Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore im...

Single nucleotide polymorphism (SNP) is the most common genetic variation among individuals. The association of SNP with individual's response to pathogens, phenotypic variations, and gene functions emphasizes the importance of sensitive and reliable SNP detection for biomedical diagnosis and therapy. To increase sensitivity, most approaches employ...

Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double-stranded DNA. Whereas single-molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C)-rich region c...

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPRn=4). Mobilities of ILPRn=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single...

The insulin linked polymorphism region (ILPR) is known to regulate transcription of the gene coding for insulin. The ILPR has guanine rich segments, suggesting that G quadruplexes may be responsible for this regulatory role. Using mechanical unfolding in a laser tweezers instrument and circular dichroism (CD) spectroscopy, we provide compelling evi...