Biology subjects

Cunningham, F. J.

Publications and source records attributed to Cunningham, F. J..

3 recordsLinked to original sources

bioRxiv2023

Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Towards this end, we introduce a generalizable platform to generate protein-SWCNT-based optical sensors and use this strategy to synthesize a hydrogen peroxide (H2O2) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. We demonstrate a concentration-dependent response to H2O2, confirm the nanosensor can image H2O2 in real-time, and assess the nanosensors selectivity for H2O2 against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.

bioengineering↗

bioRxiv2023

Mapping the Morphology of DNA on Carbon Nanotube-Based Sensors in Solution using X-ray Scattering Interferometry

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes. However, it remains unclear how the ssDNA conformation on the SWCNT surface contributes to their ultimate functionality, as observations have been constrained to computational models or experiments under dehydrated states that differ substantially from the aqueous biological environments in which the nanosensors are applied. Herein, we demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two ssDNA oligomer lengths, conformational changes as a function of ionic strength, and the mechanism of dopamine sensing for a previously established ssDNA-SWCNT nanosensor, with corresponding ab initio modeling for visualization. We show that the shorter oligomer, (GT)6, adopts a highly ordered structure of stacked rings along the SWCNT axis, compared to the longer, less periodic (GT)15 wrapping. The presence of dopamine elicits a simultaneous axial elongation and radial constriction of the ssDNA closer to the SWCNT surface. Application of XSI to probe solution-phase morphologies of nanoparticle-based tools will yield insights into sensing mechanisms and inform future design strategies for polymer-functionalized SWCNT technologies.

biophysics↗

bioRxiv2019

DNA Nanostructures Coordinate Gene Silencing in Mature Plants

Plant bioengineering may generate high yielding and stress-resistant crops amidst a changing climate and a growing global population (1-3). However, delivery of biomolecules to plants relies on Agrobacterium infection (4) or biolistic particle delivery (5), the former of which is only amenable to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall which is absent in mammalian cells and poses the dominant physical barrier to exogenous biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells, however, nanoparticle-mediated delivery remains unexplored for passive biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver small interfering RNAs (siRNAs), and effectively silence a constitutively-expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and the corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies, but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures, and details both the design parameters of importance for plant cell internalization, and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

plant biology↗