Resolving altered base-pairing of RNA modifications with DNA nanoswitches.

There are >170 naturally occurring RNA chemical modifications, with both known and unknown biological functions. Analytical methods for detecting chemical modifications and for analyzing their effects are relatively limited and have had difficulty keeping pace with the demand for RNA chemical biology and biochemistry research. Some modifications can affect the ability of RNA to hybridize with its complementary sequence or change the selectivity of base pairing. Here, we investigate the use of affinity-based DNA nanoswitches to resolve energetic differences in hybridization. We found that a single m3C modification can sufficiently destabilize hybridization to abolish a detection signal, while an s4U modification can selectively hybridize with G over A. These results establish proof of concept for using DNA nanoswitches to detect certain RNA modifications and analyzing their effects in base pairing stability and specificity.

DNA nanotechnology approaches for microRNA detection and diagnosis.

MicroRNAs are involved in the crucial processes of development and diseases and have emerged as a new class of biomarkers. The field of DNA nanotechnology has shown great promise in the creation of novel microRNA biosensors that have utility in lab-based biosensing and potential for disease diagnostics. In this Survey and Summary, we explore and review DNA nanotechnology approaches for microRNA detection, surveying the literature for microRNA detection in three main areas of DNA nanostructures: DNA tetrahedra, DNA origami, and DNA devices and motifs. We take a critical look at the reviewed approaches, advantages and disadvantages of these methods in general, and a critical comparison of specific approaches. We conclude with a brief outlook on the future of DNA nanotechnology in biosensing for microRNA and beyond.

DNA nanotechnology in the undergraduate laboratory: Analysis of molecular topology using DNA nanoswitches.

There is a disconnect between the cutting-edge research done in academic labs, such as nanotechnology, and what is taught in undergraduate labs. In the current undergraduate curriculum, very few students get a chance to do hands-on experiments in nanotechnology-related experiments most of which are through selective undergraduate research programs. In most cases, complicated synthesis procedures, expensive reagents, and requirement of specific instrumentation prevent broad adaptation of nanotechnology-based experiments to laboratory courses. DNA, being a nanoscale molecule, has recently been used in bottom-up nanotechnology with applications in sensing, nano-robotics, and computing. In this article, we propose a simple experiment involving the synthesis of a DNA nanoswitch that can change its shape from a linear "off" state to a looped "on" state in the presence of a target DNA molecule. The experiment also demonstrates the programmable topology of the looped state of the nanoswitch and its effect on gel migration. The experiment is easy to adapt in an undergraduate laboratory, requires only agarose gel electrophoresis, a minimal set-up cost for materials, and can be completed in a 3-hour time frame.

Yoctoliter thermometry for single-molecule investigations: a generic bead-on-a-tip temperature-control module.

A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.

Tuning the stability of DNA tetrahedra with base-stacking interactions.

DNA nanotechnology relies on programmable anchoring of regions of single-stranded DNA through base pair hybridization to create nanoscale objects such as polyhedra, tubes, sheets, and other desired shapes. Recent work from our lab measured energetics of base-stacking interactions and suggested that terminal stacking interactions between two adjacent strands could be an additional design parameter for DNA nanotechnology. Here, we explore that idea by creating DNA tetrahedra held together with sticky ends which contain identical base pairing interactions but different terminal stacking interactions. Testing all 16 possible combinations, we found that the melting temperature of DNA tetrahedra varied by up to 10 °C from altering a single base stack in the design while retaining a common sequence in a 6-nt sticky end. This work clearly shows that stacking design influences DNA tetrahedra stability in a substantial and predictable way. The results likely apply to other types of DNA nanostructures and suggest that terminal stacking interactions play an integral role in formation and stability of DNA nanostructures.

A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches.

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

Programmable low-cost DNA-based platform for viral RNA detection.

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures.

G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na(+), K(+), Mg(2+) and Ca(2+), promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca(2+) has the strongest stabilizing effect, followed by K(+), Mg(2+), and Na(+) in a decreasing order. The binding of K(+) to G-triplexes is accompanied by exothermic heats, and the binding of Ca(2+) with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K(+) or Ca(2+). These observations imply that stable G-triplexes may be formed under physiological conditions.