Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation

The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC “click” ligation. A study of kinetics and fidelity of replication through these artificial linkages by primer extension, PCR, and deep sequencing reveals that a subtle interplay between backbone flexibility, steric factors, and ability to hydrogen bond to the polymerase modulates rapid and accurate information decoding. Even minor phosphorothioate modifications can impair the copying process, yet some radical triazole and amide DNA backbones perform surprisingly well, indicating that the phosphate group is not essential. These findings have implications in the field of synthetic biology

Redox Capacitive Assaying of C‑Reactive Protein at a Peptide Supported Aptamer Interface

Electrochemical immunosensors offer much in the potential translation of a lab based sensing capability to a useful “real world” platform. In previous work we have introduced an impedance-derived electrochemical capacitance spectroscopic analysis as supportive of a reagentless means of reporting on analyte target capture at suitably prepared mixed-component redox-active, antibody-modified interfaces. Herein we directly integrate receptive aptamers into a redox charging peptide support in enabling a label-free low picomolar analytical assay for C-reactive protein with a sensitivity that significantly exceeds that attainable with an analogous antibody interface

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger

Optical Mie Scattering by DNA-Assembled Three-Dimensional Gold Nanoparticle Superlattice Crystals

Programmable assemblies of gold nanoparticles engineered with DNA have intriguing optical properties such as Coulomb-interaction-driven strong coupling, polaritonic response in the visible range, and ultralow dispersion dielectric response in the infrared spectral range. In this work, we demonstrate the optical Mie resonances of individual microcrystals of DNA–gold nanoparticle superlattices. Broadband hyperspectral mapping of both transmission and dark-field scattering reveal a polarization-insensitive optical response with distinct spectral features in the visible and near-infrared ranges. Experimental observations are supported by numerical simulations of the microcrystals under a resonant effective medium approximation in the regime of capacitively coupled nanoparticles. The study identifies a universal characteristic optical response which is defined by a band of multipolar Mie resonances, which only weakly depend on the crystal size and light polarization. The use of gold superlattice microcrystals as scattering materials is of interest for fields such as complex nanophotonics, thermoplasmonics, photocatalysis, sensing, and nonlinear optics