Biology subjects

Chandrasekaran, A. R.

Publications and source records attributed to Chandrasekaran, A. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Topographical features enhance DNA tetrahedron biostability

Stabilization of nucleic acid-based systems is critical for their clinical translation in biomedical applications. Here, we integrated hairpins within the edges of a DNA tetrahedron and studied the stabilization induced by these motifs against nucleases. We show that nuclease resistance of DNA tetrahedra increased with the number of hairpins in the structure, this biostability enhancement is specific to the sequences within the hairpin. Our study provides a new design approach to create nucleic acid nanostructures with inherent biostability.

bioengineering↗

bioRxiv2026

Multi-dimensional DNA nanostructures isothermally assembled in hydrated ionic liquids

DNA nanostructures can be tailored to perform a wide variety of functions, with continued interest in biological applications. Some aspects of DNA nanostructure assembly can hinder the ability of nanostructures to be useful in physiological environments. Typical assembly methods employ magnesium ions to stabilize the structure, which leave the structure susceptible to damage by nucleases in body fluids. Further, DNA nanostructure assembly typically involves a thermal annealing protocol in which DNA strands are heated in a specific buffer to a high temperature and cooled slowly at specific rates, preventing convenient encapsulation of temperature-sensitive guest molecules. In this work, we demonstrate the assembly of a wide variety of DNA nanostructures and 3D crystals in a hydrated ionic liquid (choline dihydrogen phosphate, CDHP) instead of magnesium at constant moderate temperatures, thus avoiding thermal annealing. CDHP-assembled structures show enhanced biostability against a variety of nucleases. Molecular dynamics simulations show that choline ions stabilize DNA nanostructures by a direct and close-range interaction in contrast to the predominantly water-mediated interactions of Mg2+, leading to enhanced nuclease resistance in CDHP-containing environments. CDHP-assembled structures do not affect the viability of HepG2 cells and show higher cell internalization. Overall, this work develops a potential method to construct more biostable DNA nanostructures and 3D crystals in a simple one pot process. Assembly of DNA nanostructures under isothermal conditions is desirable for scaffolding biomolecules and to reduce the need for thermal annealing instruments, allowing nanostructure preparation in low-resource settings.

biophysics↗

bioRxiv2026

Hexadecimal data encryption in paranemic crossover (PX) DNA

DNA is highly programmable and efficient for encoding information. In this work, we use the paranemic crossover (PX) DNA structures for a binary-encoded system. As substrates for data storage, we designed a combination of PX and anti-PX structures where the four strands of the PX motif are complementary to those in the anti-PX motif. To write data, we programmed encoding elements in each of the four strands of the PX and anti-PX motifs. The encoded data remains encrypted until the samples are processed at a specific temperature, when the PX and anti-PX motifs reassociate to four distinct duplexes, defined by the encoding elements and retrieved using an electrophoretic readout. We show that the encoded information is stable for several days when stored at 20 {degrees}C, 37 {degrees}C or outdoors, with the encrypted structures showing higher nuclease resistance compared to the decrypted structures. Using this strategy, we demonstrate hexadecimal encoding using a combination of 4 bits, encrypting specific words and color codes. We envision such systems could find use barcoding, secure messaging and authentication.

synthetic biology↗

bioRxiv2026

Inosine incorporation in DNA nanostructures and 3D DNA crystals

DNA nanotechnology is based on programmable base pairing, resulting in the precise construction of nanoscale structures. Sequence variability in DNA nanostructure self-assembly is achieved by the use of xeno nucleic acids, chemically modified bases and base analogs. The naturally occurring base inosine, while well studied in RNA editing, has not been used in the context of DNA nanotechnology. In this work, I demonstrate the use of inosine in DNA nanostructures, specifically by incorporating inosine within the duplex regions or junctions of a double crossover DNA motif. In strand displacement and competition assays, I show that canonical complements do not displace inosine containing strands post-assembly but dominate in product formation when competing with inosine containing strands during assembly. Finally, sticky ends with inosine base pairs enable the formation of rationally designed 3D DNA crystals based on the tensegrity triangle motif. Overall, this work shows that inosine is a useful addition to the library of sequence variations in DNA nanotechnology.

synthetic biology↗

bioRxiv2026

Aptamer-based DNA nanoswitches for multiplexed protein detection

Simple, modular platforms for detecting biologically relevant proteins are critical for applications in clinical diagnostics, healthcare, and research. Here, we have combined aptamer-based protein recognition with our conformationally-responsive DNA nanoswitches to enable simple, sensitive and specific protein detection. We demonstrate dual detection of two clinically relevant blood proteins, thrombin and VEGF as initial proof of concept.

bioengineering↗

bioRxiv2025

Paranemic cohesion of DNA molecules in different counter ions at room and physiological temperatures

Isothermal assembly allows the construction of DNA nanostructures at constant moderate temperatures. Further, assembly of DNA nanostructures in different counter ions expands their utility in various applications. In this work, we demonstrate the isothermal assembly of paranemic crossover (PX) DNA motifs in magnesium (Mg2+), calcium (Ca2+) and strontium (Sr2+) at 20 {degrees}C and 37 {degrees}C. Further, we study how differences in design affect the assembly of 4-stranded and 2-stranded PX molecules.

synthetic biology↗

bioRxiv2025

Controlled reassociation of multistranded, polycrossover DNA molecules into double helices

Shape-changing DNA nanostructures have found applications in biosensing, drug delivery, cell modulation and data storage. A key aspect of this reconfiguration is the interaction of DNA nanostructures with other biomolecules or chemical stimuli such as pH and ionic conditions. Sequence-based nanostructure reconfiguration is largely achieved by strand displacement which is based on single stranded toeholds. In this work, we use sequence and temperature-controlled reassociation of one type of a DNA nanostructure into another structure. We demonstrate this strategy using the paranemic crossover (PX) DNA, a four-stranded structure with two adjacent double helical domains connected by six strand crossovers. In the presence of an anti-PX structure that is composed of strands that are each complementary to those in PX DNA, the structures reassociate at specific temperatures to form duplexes. Using the denaturing agent formamide, we decreased the temperature required for this reassociation. We extend the strategy to other polycrossover DNA molecules such as a double crossover motif (2 crossovers) and a juxtaposed DNA motif (4 crossovers), showing controlled reassociation of different DNA motifs into duplexes. Our study highlights the potential for DNA motifs to function as switchable molecular systems, offering new insights for responsive DNA-based materials and devices.

synthetic biology↗

bioRxiv2025

Peptide nucleic acid (PNA) and DNA hybrid three-way junctions and mesojunctions

Three-way junctions are simple and fundamental structural motifs that impart the typical branching property in most DNA nanostructures. While conventional three-way DNA junctions are well-known, mesojunctions are relatively unexplored. Here, we report the synthesis of peptide nucleic acid (PNA)/DNA hybrid three-way conventional and mesojunctions, containing a 14 bp duplex DNA arm and two PNA/DNA hybrid arms. We show that the PNA/DNA mesojunction can be assembled in magnesium-free buffers containing low concentrations of calcium or sodium salts. PNA/DNA hybrid junctions and mesojunctions showed higher thermal stability compared to the DNA versions. Further, PNA/DNA hybrid junctions assembled in sodium exhibited higher nuclease resistance against DNase I. Our results pave the way for PNA/DNA hybrid three-way junctions to be integrated into complex DNA nanostructure designs to improve their structural and enzymatic stability.

synthetic biology↗

bioRxiv2025

Mechanistic Insights into Crossover-Dependent Stability and Exceptional Resistance of PX/JX Nanostructures to DNase I using Enhanced Sampling

Designing biostable DNA nanocarriers for precise therapeutic delivery remains a key challenge in DNA nanotechnology due to susceptibility to nuclease degradation. Multi-stranded DNA nanostructures, such as Paranemic crossover (PX) DNA, show enhanced biostability compared to native duplex DNA due to their unique topology. However, the molecular origin of their exceptional nuclease resistance is still unknown. Using atomistic MD simulation and enhanced sampling, we uncover the molecular origins of PX-DNAs superior resistance over JX-DNA and dsDNA. Our findings reveal that PX-DNAs six crossover points induce an overtwisted helix and narrower minor groove, leading to reduced DNase I binding affinity ([~] +5 kcal/mol for PX-DNA vs. [~] -17 kcal/mol for dsDNA). Mechanical properties, such as the stretch modulus ({gamma}G), confirm enhanced structural rigidity of PX-DNA (4805 pN), while residence time calculations using{tau} -RAMD further highlight shorter residence time of DNase I nuclease on PX-DNA. This is the first theoretical study to explore crossover-dependent biostability, mechanical properties, and residence time of PX/JX nanostructures in the presence of a nuclease using enhanced sampling. Our findings highlight that strategically positioned crossover points can regulate DNA stability against nuclease degradation at the nanoscale and lead to better design of biostable DNA nanostructures for medicinal applications.

biophysics↗

bioRxiv2024

Influence of metal ions on the isothermal self-assembly of DNA nanostructures

DNA nanostructures are typically assembled by thermal annealing in buffers containing magnesium. We demonstrate the assembly of DNA nanostructures at constant temperatures ranging from 4 {degrees}C to 50 {degrees}C in solutions containing different metal ions. The choice of metal ions and the assembly temperature influence the isothermal assembly of several DNA motifs and designed three-dimensional DNA crystals. Molecular dynamics simulations show more fluctuations of the DNA structure in select monovalent ions (Na+ and K+) compared to divalent ions (Mg2+ and Ca2+). A key highlight is the successful assembly of DNA motifs in nickel-containing buffer at temperatures below 40 {degrees}C, otherwise unachievable at higher temperatures, or using an annealing protocol. DNA nanostructures isothermally assembled in different ions do not affect the viability of fibroblasts, myoblasts, and myotubes and or the immune response in myoblasts. The use of ions other than the typically-used magnesium holds key potential in biological and materials science applications that require minimal amounts of magnesium.

bioengineering↗

bioRxiv2024

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 the 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 that 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 {degrees}C from altering a single base stack in the design. These results can inform stacking design to control DNA tetrahedra stability in a substantial and predictable way. To that end, we show that a 4 bp sticky end with weak terminal stacking does not form stable tetrahedra, while strengthening the stacks confers high stability with a 46.8 {+/-} 1.2 {degrees}C melting temperature, comparable to a 6 bp sticky end with weak stacking (49.7 {+/-} 2.9 {degrees}C). 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.

bioengineering↗

bioRxiv2023

Unusual Structural Properties 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.

bioengineering↗

bioRxiv2023

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 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.

synthetic biology↗

bioRxiv2023

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.

synthetic biology↗

bioRxiv2022

Quantifying individual base stacking energies with the Centrifuge Force Microscope

Base stacking interactions between adjacent bases in DNA and RNA are known to be important for many biological processes, for drug development, and in other biotechnology applications. While previous work has estimated base stacking energies between pairs of bases, the individual contributions of each base to the stacking interaction has remained unknown. Here, we developed a novel methodology using a custom Centrifuge Force Microscope to perform high-throughput single molecule experiments to measure base stacking energies between individual 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 -0.4 {+/-} 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA bases had no measurable effect, but a fluorophore modification reduced stacking energy. The implications of the work are demonstrated with three applications. We experimentally show that base stacking design can influence assembly and stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and determine accuracy of force fields in molecular dynamics (MD) simulations. Our results provide new insights into fundamental DNA interactions that are critical in biology and can inform rational design in diverse biotechnology applications.

biophysics↗

bioRxiv2021

Detection of Alzheimer's associated microRNAs using a DNA-based smart reagent

Alzheimers disease (AD) is the most common neurodegenerative disorder, with significant research efforts devoted to identifying new biomarkers for clinical diagnosis and treatment. MicroRNAs have emerged as likely disease regulators and biomarkers for AD, now implicated as having roles in several biological processes related to progression of the disease. In this work, we use the miRacles assay (microRNA activated conditional looping of engineered switches) for single-step detection of AD-related microRNAs. The technology is based on conformationally responsive DNA nanoswitches that loop upon recognition of a target microRNA and report their on/off status through an electrophoretic readout. Unlike many other methods, our approach directly detects native microRNAs without amplification or labeling, eliminating the need for expensive enzymes, reagents, and equipment. We used this assay to screen for AD-related microRNAs, demonstrate specificity within a microRNA family, sensitivity of [~] 8 fM, and multiplexing capability to simultaneously detect four microRNA targets. Toward clinical use, we provide proof-of-concept detection and quantifiable dysregulation of specific microRNAs from total RNA extracts derived from healthy and AD brain samples. In the context of AD, this "smart reagent" could facilitate biomarker discovery, accelerate efforts to understand the role of microRNAs in AD, and have clinical potential as a diagnostic or monitoring tool for validated biomarkers.

bioengineering↗

bioRxiv2020

DNA nanoswitch barcodes for multiplexed biomarker profiling

The detection of molecular biomarkers plays a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. The ability to detect 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. One promising approach is the use of dynamic DNA nanostructures that can respond to molecular biomarkers, which have recently been used in a variety of biosensing strategies. In this work, we report the use of programmable DNA nanoswitches for the multiplexed detection of up to 6 biomarkers within a single pot through the use of a barcoded gel-based readout. We demonstrate the barcoding capability using gene fragments that correspond to 6 different diseases, with each fragment or combination of fragments producing a unique barcode signature. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules - 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 (PSA). This strategy holds promise in clinical diagnostics for profiling complex and diverse biomarker panels.

bioengineering↗

bioRxiv2020

Single species RNA purification with DNA nanoswitches

We report a novel method to purify individual RNA species using programmable DNA nanoswitches to facilitate RNA capture, release, and isolation. We validate sequence-based purification of microRNA, rRNA, and an mRNA fragment from total RNA, demonstrate multiplexing with two different RNA species, and show an application with downstream LC/MS analysis to identify RNA modifications. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption.Competing Interest StatementLZ, ARC, and KH have intellectual property related to DNA nanoswitches. All other authors declare no competing interests.View Full Text

molecular biology↗