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.

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.

Single-Molecule Analysis of Nanocircle-Embedded I-Motifs under Crowding.

Cytosine (C)-rich regions of single-stranded DNA or RNA can fold into a tetraplex structure called i-motifs, which are typically stable under acidic pHs due to the need for protons to stabilize C-C interactions. While new studies have shown evidence for the formation of i-motifs at neutral and even physiological pH, it is not clear whether i-motifs can stably form in cells where DNA experiences topological constraint and crowding. Similarly, several studies have shown that a molecularly crowded environment promotes the formation of i-motifs at physiological pH; however, whether the intracellular crowding counteracts the topological destabilization of i-motifs is yet to be investigated. In this manuscript, using fluorescence resonance energy transfer (FRET)-based single-molecule analyses of human telomeric (hTel) i-motifs embedded in nanocircles as a proof-of-concept platform, we investigated the overall effects of crowding and topological constraint on the i-motif behavior. The smFRET analysis of the nanoassembly showed that the i-motif remains folded at pH 5.5 but unfolds at higher pHs. However, in the presence of a crowder (30% PEG 6000), i-motifs are formed at physiological pH overcoming the topological constraint imposed by the DNA nanocircles. Analysis of FRET-time traces show that the hTel sequence primarily assumes the folded state at pH ≤7.0 under crowding, but it undergoes slow conformational transitions between the folded and unfolded states at physiological pH. Our demonstration that the i-motif can form under cell-mimic crowding and topologically constrained environments may provide new insights into the potential biological roles of i-motifs and also into the design and development of i-motif-based biosensors, therapy, and other nanotechnological applications.

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.

Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope.

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 region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.