Biology subjects

Rajwar, A.

Publications and source records attributed to Rajwar, A..

6 recordsLinked to original sources

bioRxiv2026

Evolutionary selection of DNA nanostructures for cellular uptake

DNA nanotechnology offers precise, biocompatible structures with strong potential for targeted drug delivery, yet current discovery approaches rely on testing individual designs, limiting exploration of structural diversity. Here, we introduce an evolutionary selection strategy that screens large libraries of DNA nanostructures, each folded from a single-stranded structure genome compatible with amplification and sequencing. Cellular internalization is used as the selection pressure: libraries are incubated with mammalian cells, internalized structures are recovered from lysates, and the process is iterated across multiple rounds in HEK293T and RAW264.7 cells. High-throughput sequencing of recovered structure genomes reveals cell-type-specific enrichment patterns, enabling the identification of individual nanostructures with preferential uptake. Selected candidates were synthesized and evaluated as purified structures, confirming differential internalization by quantitative flow cytometry and microscopy. Turning DNA nanostructure discovery into a selection-based process, could enable high-throughput exploration of structural diversity and provide an alternative route to identify nanostructures with cell-specific uptake properties for biomedical applications.

bioengineering↗

bioRxiv2025

cONcat: Computational reconstruction of concatenated fragments from long Oxford Nanopore reads

Synthetic combinatorial DNA libraries are widely used to produce protein variants, optimize binders, and for high throughput studies of protein - DNA interactions. The libraries can be made by researchers or vendors and high-throughput sequencing is used for both quality control and to study the outcome of selection experiments. Oxford nanopore sequencing (ONT) is well suited to this as it allows for long read lengths and can be done rapidly with low-cost instrumentation. However, it suffers from a lower overall read accuracy and an uneven error profile. No current bioinformatics tools are well suited to the challenge of deducing the composition and order of constituent members of combinatorial libraries from ONT reads. We introduce cONcat, an algorithm to identify the makeup of concatenated DNA fragments in a set of ONT sequencing reads from a pool of known fragments. cONcat uses the edit distance-based recursive covering algorithm for finding the best possible matchings between the fragments and the reads. In our experiments on simulated and experimental data, cONcat could accurately detect the correct fragment coverings given the short fragment sizes (< 20bp) and the sequencing errors present in ONT reads. However, we find that the high error rates in the start of ONT reads make it challenging to get confident coverage there, inferring a need for experimental strategies to avoid key sequence information in the start of reads.

bioinformatics↗

bioRxiv2024

Validation of DoriVac (DNA origami vaccine) efficacy in a metastatic melanoma model

Metastatic cancer, particularly metastatic melanoma, poses a significant therapeutic challenge due to its resistance to standard treatments and low five-year survival rate of 20-27%. Current therapies show limited success, highlighting the urgent need for novel interventions. Recent advances in cancer vaccine research show great promise in reducing disease recurrence, but these approaches still face challenges pertaining to antigen selection, ease of production, and programmability. To address some of these challenges, we have adapted the DoriVac platform to serve as a cancer vaccine against metastatic melanoma. DoriVac is a DNA origami-based vaccine platform that allows for the codelivery of antigens of choice and the CpG immune adjuvant at an optimal nanospacing to promote Th1 immune polarization. In our study, we observed a significant reduction in lung tumor nodules for mice treated with DoriVac in both the B16OVA and B16F10 melanoma mouse models. DoriVac also appears to be a safe and effective treatment, with no anti-drug antibodies, as indicated by lower anti-dsDNA levels. Importantly, we observed an amplified effect when DoriVac was combined with PD-L1 immune checkpoint blockade, leading to an even greater reduction in metastatic lung tumor nodules. Our results also indicate an increased activation of antigen presenting cells, NK cells, CD4+ T cells and CD8+ T cells. These findings suggest that DoriVac, particularly in combination with the PD-L1 immune checkpoint blockade, can serve as a promising immunotherapy against metastatic cancer.

synthetic biology↗

bioRxiv2024

Cargo quantification of functionalized DNA origami for therapeutic application

In recent years, notable advances in nanotechnology-based drug delivery have emerged. A particularly promising platform in this field is DNA origami-based nanoparticles, which offer highly programmable surfaces, providing precise control over the nanoscale spacing and stoichiometry of various cargo. These versatile particles are finding diverse applications ranging from basic molecular biology to diagnostics and therapeutics. This growing interest creates the need for effective methods to quantify cargo on DNA origami nanoparticles. Our study consolidates several previously validated methods focusing on gel-based and fluorescence-based techniques, including multiplexed quantification of protein, peptide, and nucleic acid cargo on these nanoparticles. This work may serve as a valuable resource for groups researchers keen on utilizing DNA origami-based nanoparticles in therapeutic applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/609963v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1703793org.highwire.dtl.DTLVardef@50b368org.highwire.dtl.DTLVardef@327a3corg.highwire.dtl.DTLVardef@1f732b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine nanotechnology, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific heptad repeat 2 (HR2) peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naive mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular and humoral immune responses in human cells. Moreover, DoriVac bearing full-length SARS-CoV-2 spike protein achieved immune responses comparable to current mRNA vaccine platforms while potentially reducing storage constraints. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities, underscoring its potential utility in addressing future pandemics.

immunology↗

bioRxiv2021

Ultrasound exposure enhances cellular uptake of nanoscale cargos

DNA nanotechnology utilizes DNA as a structural molecule to design palette of nanostructures with different shapes and sizes. DNA nanocages have demonstrated significant potential for drug delivery. Therefore, enhancing the delivery of DNA nanocages into cells can improve their efficacy as drug delivery agents. Numerous studies have reported the effects of ultrasound for enhancing drug delivery across biological barriers. The mechanical bioeffects caused by cell-ultrasound interaction can cause sonoporation, leading to enhanced uptake of drugs, nanoparticles, and chemotherapeutic agents through membranes. Whether ultrasound exposure can enhance the delivery of DNA nanocages has not been explored, which is the focus of this study. Specifically, we investigated the effects of ultrasound on the cellular uptake of propidium Iodide, fluorescent dextrans, and DNA nanostructures). We provide evidence of modulation of pore formation in the cell membrane by ultrasound by studying the intracellular uptake of the impermeable dye, propidium iodide. Treatment of cells with low amplitudes of ultrasound enhanced the uptake of different sizes of dextrans and DNA based nanodevices. These findings could serve as the foundation for further development ultrasound-enabled DNA nanostructure delivery and for specific understanding of underlying biological mechanisms of interaction between ultrasound parameters and cellular components; the knowledge that can be further explored for potential biological and biomedical applications.

bioengineering↗