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.

Barium Concentration-Dependent Anomalous Electrophoresis of Synthetic DNA Motifs.

The structural integrity, assembly yield, and biostability of DNA nanostructures are influenced by the metal ions used to construct them. Although high (>10 mM) concentrations of divalent ions are often preferred for assembling DNA nanostructures, the range of ion concentrations and the composition of the assembly products vary for different assembly conditions. Here, we examined the unique ability of Ba2+ to retard double crossover DNA motifs by forming a low mobility species, whose mobility on the gel is determined by the concentration ratio of DNA and Ba2+. The formation of this electrophoretically retarded species is promoted by divalent ions such as Mg2+, Ca2+, and Sr2+ when combined with Ba2+ but not on their own, while monovalent ions such as Na+, K+, and Li+ do not have any effect on this phenomenon. Our results highlight the complex interplay between the metal ions and DNA self-assembly and could inform the design of DNA nanostructures for applications that expose them to multiple ions at high concentrations.

The unusual structural properties and potential biological relevance of switchback DNA.

Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

A DNA rotary nanodevice operated by enzyme-initiated strand resetting.

DNA nanostructures that respond to external stimuli have found applications in several areas such as biosensing, drug delivery and molecular computation. The use of different types of stimuli in a single operation provides another layer of control for the reconfiguration of nucleic acid nanostructures. This work demonstrates the use of a ribonuclease to "unset" a nucleic acid nanodevice based on the paranemic crossover (PX) DNA and specific DNA inputs to "reset" the structure into a juxtaposed DNA (JX2) configuration, resulting in a 180° rotation of the helical domains. Such operations would be useful in translational applications where DNA nanostructures can be designed to reconfigure on the basis of more than one stimulus.

siRNA-loaded DNA nanostructures restore endothelial leakiness.

This article highlights the recent work of Qian, Leong, Wang et al. (Nanoscale Horiz., 2023, 8, 270, https://doi.org/10.1039/D2NH00348A) on the use of programmable DNA self-assemblies to restore endothelial leakiness.

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.

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.

Introduction to emerging concepts in nucleic acids: structures, functions and applications.

An introduction to the Nanoscale, Nanoscale Advances and Physical Chemistry Chemical Physics (PCCP) themed collection on DNA and RNA nanotechnology, featuring a selection of excellent articles that highlight the potential of nucleic acids for various applications.

Fluorometric Determination of DNA Nanostructure Biostability.

The analysis and improvement of DNA nanostructure biostability is one of the keys areas of progress needed in DNA nanotechnology applications. Here, we present a plate-compatible fluorometric assay for measuring DNA nanostructure biostability using the common intercalator ethidium bromide. We demonstrate the assay by testing the biostability of duplex DNA, a double crossover DNA motif, and a DNA origami nanostructure against different nucleases and in fetal bovine serum. This method scales well to measure a large number of samples using a plate reader and can complement existing methods for assessing and developing robust DNA nanostructures.

Self-Assembly of DNA Nanostructures in Different Cations.

The programmable nature of DNA allows the construction of custom-designed static and dynamic nanostructures, and assembly conditions typically require high concentrations of magnesium ions that restricts their applications. In other solution conditions tested for DNA nanostructure assembly, only a limited set of divalent and monovalent ions are used so far (typically Mg2+ and Na+ ). Here, we investigate the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes: a double-crossover motif (76 bp), a three-point-star motif (~134 bp), a DNA tetrahedron (534 bp) and a DNA origami triangle (7221 bp). We show successful assembly of a majority of these structures in Ca2+ , Ba2+ , Na+ , K+ and Li+ and provide quantified assembly yields using gel electrophoresis and visual confirmation of a DNA origami triangle using atomic force microscopy. We further show that structures assembled in monovalent ions (Na+ , K+ and Li+ ) exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions (Mg2+ , Ca2+ and Ba2+ ). Our work presents new assembly conditions for a wide range of DNA nanostructures with enhanced biostability.

Self-assembly of DNA nanostructures in different cations.

The programmable nature of DNA allows the construction of custom-designed static and dynamic nanostructures, and assembly conditions typically require high concentrations of magnesium ions which restricts their applications. In other solution conditions tested for DNA nanostructure assembly, only a limited set of divalent and monovalent ions have been used so far (typically Mg 2+ and Na + ). Here, we investigate the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes: a double-crossover motif (76 bp), a three-point-star motif (∼134 bp), a DNA tetrahedron (534 bp) and a DNA origami triangle (7221 bp). We show successful assembly of a majority of these structures in Ca 2+ , Ba 2+ , Na + , K + and Li + and provide quantified assembly yields using gel electrophoresis and visual confirmation of a DNA origami triangle using atomic force microscopy. We further show that structures assembled in monovalent ions (Na + , K + and Li + ) exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions (Mg 2+ , Ca 2+ and Ba 2+ ). Our work presents new assembly conditions for a wide range of DNA nanostructures with enhanced biostability.

Caffeine-induced release of small molecules from DNA nanostructures.

Several planar aromatic molecules are known to intercalate between base pairs of double-stranded DNA. This mode of interaction has been used to stain DNA as well as to load drug molecules onto DNA-based nanostructures. Some small molecules are also known to induce deintercalation in double-stranded DNA, one such molecule being caffeine. Here, we compared the ability of caffeine to cause deintercalation of ethidium bromide, a representative DNA intercalator, from duplex DNA and three DNA motifs of increasing structural complexity (four-way junction, double crossover motif, and DNA tensegrity triangle). We found that caffeine impedes the binding of ethidium bromide in all these structures to the same extent, with some differences in deintercalation profiles. Our results can be useful in the design of DNA nanocarriers for intercalating drugs, where drug release can be chemically stimulated by other small molecules.

DNA nanotechnology in the undergraduate laboratory: Electrophoretic analysis of DNA nanostructure biostability.

The field of DNA nanotechnology has grown rapidly in the last decade and has expanded to multiple laboratories. While lectures in DNA nanotechnology have been introduced in some institutions, laboratory components at the undergraduate level are still lacking. Undergraduate students predominantly learn about DNA nanotechnology through their involvement as interns in research laboratories. The DNA nanostructure biostability analysis experiment presented here can be used as a hands-on introductory laboratory exercise for discussing concepts in DNA nanotechnology in an undergraduate setting. This experiment discusses biostability, gel electrophoresis and quantitative analysis of nuclease degradation of a model DNA nanostructure, the paranemic crossover (PX) DNA motif. The experiment can be performed in a chemistry, biology or a biochemistry laboratory with minimal costs and can be adapted in undergraduate institutions using the instructor and student manuals provided here. Laboratory courses based on cutting edge research not only provide students a direct hands-on approach to the subject, but can also increase undergraduate student participation in research. Moreover, laboratory courses that reflect the increasingly multidisciplinary nature of research add value to undergraduate education.

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.

The Formation and Displacement of Ordered DNA Triplexes in Self-Assembled Three-Dimensional DNA Crystals.

Reconfigurable structures engineered through DNA hybridization and self-assembly offer both structural and dynamic applications in nanotechnology. Here, we have demonstrated that strand displacement of triplex-forming oligonucleotides (TFOs) can be translated to a robust macroscopic DNA crystal by coloring the crystals with covalently attached fluorescent dyes. We show that three different types of triplex strand displacement are feasible within the DNA crystals and the bound TFOs can be removed and/or replaced by (a) changing the pH from 5 to 7, (b) the addition of the Watson-Crick complement to a TFO containing a short toehold, and (c) the addition of a longer TFO that uses the duplex edge as a toehold. We have also proved by X-ray diffraction that the structure of the crystals remains as designed in the presence of the TFOs.

Encoding, Decoding, and Rendering Information in DNA Nanoswitch Libraries.

DNA-based construction allows the creation of molecular devices that are useful in information storage and processing. Here, we combine the programmability of DNA nanoswitches and stimuli-responsive conformational changes to demonstrate information encoding and graphical readout using gel electrophoresis. We encoded information as 5-bit binary codes for alphanumeric characters using a combination of DNA and RNA inputs that can be decoded using molecular stimuli such as a ribonuclease. We also show that a similar strategy can be used for graphical visual readout of alphabets on an agarose gel, information that is encoded by nucleic acids and decoded by a ribonuclease. Our method of information encoding and processing could be combined with DNA actuation for molecular computation and diagnostics that require a nonarbitrary visual readout.

Toehold clipping: A mechanism for remote control of DNA strand displacement.

The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation.

Purification of Self-Assembled DNA Tetrahedra Using Gel Electrophoresis.

DNA nanostructures have found applications in a variety of fields such as biosensing, drug delivery, cellular imaging, and computation. Several of these applications require purification of the DNA nanostructures once they are assembled. Gel electrophoresis-based purification of DNA nanostructures is one of the methods used for this purpose. Here, we describe a step-by-step protocol for a gel-based method to purify self-assembled DNA tetrahedra. With further optimization, this method could also be adapted for other DNA nanostructures. © 2022 Wiley Periodicals LLC. Basic Protocol: Purification of self-assembled DNA tetrahedra.