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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.

 

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.

 

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. 

Custom‐built DNA nanostructures are now used in applications such as biosensing, molecular computation, biomolecular analysis, and drug delivery. While the functionality and biocompatibility of DNA makes DNA nanostructures useful in such applications, the field faces a challenge in making biostable DNA nanostructures. Being a natural material, DNA is most suited for biological applications, but is also easily degraded by nucleases. Several methods have been employed to study the nuclease degradation rates and enhancement of nuclease resistance. This protocol describes the use of gel electrophoresis to analyze the extent of nuclease degradation of DNA nanostructures and to report degradation times, kinetics of nuclease digestion, and evaluation of biostability enhancement factors. © 2020 Wiley Periodicals LLC.

Basic Protocol: Timed analysis of nuclease degradation of DNA nanostructures

Support Protocol: Calculating biostability enhancement factors

 

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.