DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling.

Molecular biomarkers play a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. Detecting multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. Here, we report programmable DNA nanoswitches for multiplexed detection of up to 6 biomarkers at once with each combination of biomarkers producing a unique barcode signature among 64 possibilities. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules, for example, DNA, RNA, antibody, and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen.

Orthogonal Control of DNA Nanoswitches with Mixed Physical and Biochemical Cues.

Nanoscale devices that can respond to external stimuli have potential applications in drug delivery, biosensing, and molecular computation. Construction using DNA has provided many such devices that can respond to cues such as nucleic acids, proteins, pH, light, or temperature. However, simultaneous control of molecular devices is still limited. Here, we present orthogonal control of DNA nanoswitches using physical (light) and biochemical (enzyme and nucleic acid) triggers. Each one of these triggers controls the reconfiguration of specific nanoswitches from locked to open states within a mixture and can be used in parallel to control a combination of nanoswitches. Such dynamic control over nanoscale devices allows the incorporation of tunable portions within larger structures as well as spatiotemporal control of DNA nanostructures.

Exceptional Nuclease Resistance of Paranemic Crossover (PX) DNA and Crossover-Dependent Biostability of DNA Motifs.

Nanometer-sized features and molecular recognition properties make DNA a useful material for nanoscale construction, but degradation in biological fluids poses a considerable roadblock to biomedical applications of DNA nanotechnology. Here, we report the remarkable biostability of a multistranded motif called paranemic crossover (PX) DNA. Compared to double stranded DNA, PX DNA has dramatically enhanced (sometimes >1000 fold) resistance to degradation by four different nucleases, bovine and human serum, and human urine. We trace the cause of PX's biostability to DNA crossovers, showing a continuum of protection that scales with the number of crossovers. These results suggest that enhanced biostability can be engineered into DNA nanostructures by adopting PX-based architectures or by strategic crossover placement.

The role of size in biostability of DNA tetrahedra.

The potential for using DNA nanostructures for drug delivery applications requires understanding and ideally tuning their biostability. Here we investigate how biological degradation varies with size of a DNA nanostructure. We designed DNA tetrahedra of three edge lengths ranging from 13 to 20 bp and analyzed nuclease resistance for two nucleases and biostability in fetal bovine serum. We found that DNase I had similar digestion rates across sizes but appeared to incompletely digest the smallest tetrahedron, while T5 exonuclease was notably slower to digest the largest tetrahedron. In fetal bovine serum, the 20 bp tetrahedron was degraded ~four times faster than the 13 bp. These results show that DNA nanostructure size can influence nuclease degradation, but suggest a complex relationship that is nuclease specific.

The role of size in biostability of DNA tetrahedra.

The potential for using DNA nanostructures for drug delivery applications requires understanding and ideally tuning their biostability. Here we investigate how biological degradation varies with size of a DNA nanostructure. We designed DNA tetrahedra of three edge lengths ranging from 13 to 20 bp and analyzed nuclease resistance for two nucleases and biostability in fetal bovine serum. We found that DNase I had similar digestion rates across sizes but appeared to incompletely digest the smallest tetrahedron, while T5 exonuclease was notably slower to digest the largest tetrahedron. In fetal bovine serum, the 20 bp tetrahedron was degraded four times faster than the 13 bp. These results show that DNA nanostructure size can influence nuclease degradation, but suggest a complex relationship that is nuclease specific.

High-throughput single-molecule quantification of individual base stacking energies in nucleic acids.

Base stacking interactions between adjacent bases in DNA and RNA are important for many biological processes and in biotechnology applications. Previous work has estimated stacking energies between pairs of bases, but contributions of individual bases has remained unknown. Here, we use a Centrifuge Force Microscope for high-throughput single molecule experiments to measure stacking energies between adjacent bases. We found stacking energies strongest between purines (G|A at -2.3 ± 0.2 kcal/mol) and weakest between pyrimidines (C|T at -0.5 ± 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA nucleotides had no measurable effect, but a fluorophore modification reduced stacking energy. We experimentally show that base stacking can influence stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and assess accuracy of force fields in molecular dynamics simulations. Our results provide insights into fundamental DNA interactions that are critical in biology and can inform design in biotechnology applications.

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.

Sequence-selective purification of biological RNAs using DNA nanoswitches.

Nucleic acid purification is a critical aspect of biomedical research and a multibillion-dollar industry. Here we establish sequence-selective RNA capture, release, and isolation using conformationally responsive DNA nanoswitches. We validate purification of specific RNAs ranging in size from 22 to 401 nt with up to 75% recovery and 99.98% purity in a benchtop process with minimal expense and equipment. Our method compared favorably with bead-based extraction of an endogenous microRNA from cellular total RNA, and can be programmed for multiplexed purification of multiple individual RNA targets from one sample. Coupling our approach with downstream LC/MS, we analyzed RNA modifications in 5.8S ribosomal RNA, and found 2'-O-methylguanosine, 2'-O-methyluridine, and pseudouridine in a ratio of ~1:7:22. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption, and the versatility of the approach provides opportunities to expand the strategy to other biomolecules.

Hybrid DNA/RNA nanostructures with 2'-5' linkages.

Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2'-5' linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2'-5' linkages. We synthesized and incorporated RNA strands with 2'-5' linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2'-5' linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions.