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-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

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

Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

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 molecule studies using optical tweezers showed that the two quadruplexes in the ILPRn=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPRn=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPRn=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPRn=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR

Intramolecular Folding in Human ILPR Fragment with Three C-Rich Repeats

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-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures

Invited Review Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014

ABSTRACT: Four days after the announcement of the 2014 Nobe

Mechanical stability evaluation of i-motif and G-quadruplex structures under diverse circumstances

G-quadruplex is the most widely known four-stranded nucleic acid structure which has shown to alter gene regulation both in vitro and in vivo. Under certain conditions, another four-stranded structure, i-motif, is also formed in the strand complementary to the G-quadruplex forming sequence. Recent studies suggest gene regulatory roles for the i-motif structure as well. Although there is substantial understanding on the folding topology of G-quadruplex and i-motif structures, their mechanical stability which determines the interaction with motor proteins, such as DNA/RNA polymerases, are poorly studied. Since DNA exists as a double stranded form in vivo, the investigation of i-motif becomes highly important to fully understand the biological functions of G-quadruplexes. Using laser tweezers based single-molecule study, we investigated the mechanical stability of an i-motif structure in the predominant variant of human ILPR fragment (5'-TGTC4ACAC4TGTC4ACAC4TGT). In addition, we have shown that a partially folded structure composed of only three tandem C-rich repeats coexists with the i-motif. Both structures share similar unfolding forces of 22-26 pN. Discovery of stable structures in less than four C-rich repeats suggested that the structure can serve as an intermediate during the i-motif folding/unfolding pathway. Using chemical footprinting and single-molecule approaches, we show that a dsDNA fragment in ILPR, 5'-(ACAG4TGTG4ACAG4TGTG4ACA), can fold into G-quadruplex or i-motif structure under specific conditions. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif, changes in free energy of unfolding provided compelling evidence that only one species is present in each dsDNA. Based on this observation, we propose that G-quadruplex and i-motif are mutually exclusive in human ILPR. Furthermore, we show that these two species have an unfolding force >17 pN. From mechanical perspective, this could justify the regulatory role a DNA tetraplex may play in the expression of human insulin inside cells in which dsDNA is the predominate form

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-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 <i>Escherichia coli</i> 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