Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of Protein N-Glycosylation.

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation.

A Discovery with Potential to Revitalize Hammerhead Ribozyme Therapeutics for Treatment of Inherited Retinal Degenerations.

Hammerhead ribozymes (hhRzs), RNA enzymes capable of site-specific cleavage of arbitrary target mRNAs, have faced significant hurdles in development and optimization as gene therapeutics for clinical translation. Chemical and biological barriers must be overcome to realize an effective therapeutic. A new Facilitated ribozyme has been identified with greatly enhanced kinetic properties that lead new insight on the capacity of ribozymes to target mutant genes to treat inherited retinal degenerations.

Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.

Purpose: To systematically evaluate human rod opsin () mRNA for potential target sites sensitive to posttranscriptional gene silencing (PTGS) by hammerhead ribozyme (hhRz) or RNA interference (RNAi) in human cells. To develop a comprehensive strategy to identify and optimize lead candidate agents for PTGS gene therapeutics.

Methods: In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polymerase chain reaction (RT-PCR) were used to map accessibility in full-length transcripts.

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression.

High-resolution microstrip NMR detectors for subnanoliter samples.

We present the numerical optimization and experimental characterization of two microstrip-based nuclear magnetic resonance (NMR) detectors. The first detector, introduced in our previous work, was a flat wire detector with a strip resting on a substrate, and the second detector was created by adding a ground plane on top of the strip conductor, separated by a sample-carrying capillary and a thin layer of insulator. The dimensional parameters of the detectors were optimized using numerical simulations with regards to radio frequency (RF) sensitivity and homogeneity, with particular attention given to the effect of the ground plane.

Toward high-resolution NMR spectroscopy of microscopic liquid samples.

A longstanding limitation of high-resolution NMR spectroscopy is the requirement for samples to have macroscopic dimensions. Commercial probes, for example, are designed for volumes of at least 5 μL, in spite of decades of work directed toward the goal of miniaturization. Progress in miniaturizing inductive detectors has been limited by a perceived need to meet two technical requirements: (1) minimal separation between the sample and the detector, which is essential for sensitivity, and (2) near-perfect magnetic-field homogeneity at the sample, which is typically needed for spectral resolution.

A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets.

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease.

A Novel, Real-Time, In Vivo Mouse Retinal Imaging System.

Purpose: To develop an efficient, low-cost instrument for robust real-time imaging of the mouse retina in vivo, and assess system capabilities by evaluating various animal models.

Methods: Following multiple disappointing attempts to visualize the mouse retina during a subretinal injection using commercially available systems, we identified the key limitation to be inadequate illumination due to off axis illumination and poor optical train optimization. Therefore, we designed a paraxial illumination system for Greenough-type stereo dissecting microscope incorporating an optimized optical launch and an efficiently coupled fiber optic delivery system.

Parahydrogen-induced polarization at zero magnetic field.

We use symmetry arguments and simple model systems to describe the conversion of the singlet state of parahydrogen into an oscillating sample magnetization at zero magnetic field. During an initial period of free evolution governed by the scalar-coupling Hamiltonian HJ, the singlet state is converted into scalar spin order involving spins throughout the molecule. A short dc pulse along the z axis rotates the transverse spin components of nuclear species I and S through different angles, converting a portion of the scalar order into vector order.

Multiplets at zero magnetic field: the geometry of zero-field NMR.

For liquid samples at Earth's field or below, nuclear-spin motion within scalar-coupled networks yields multiplets as a spectroscopic signature. In weak fields, the structure of the multiplets depends on the magnitude of the Zeeman interaction relative to the scalar couplings; in Earth's field, for example, heteronuclear couplings are truncated by fast precession at distinct Larmor frequencies. At zero field, weak scalar couplings are truncated by the relatively fast evolution associated with strong scalar couplings, and the truncated interactions can be described geometrically.

High-resolution zero-field NMR J-spectroscopy of aromatic compounds.

We report the acquisition and interpretation of nuclear magnetic resonance (NMR) J-spectra at zero magnetic field for a series of benzene derivatives, demonstrating the analytical capabilities of zero-field NMR. The zeroth-order spectral patterns do not overlap, which allows for straightforward determination of the spin interactions of substituent functional groups. Higher-order effects cause additional line splittings, revealing additional molecular information.

Zero-field NMR enhanced by parahydrogen in reversible exchange.

We have recently demonstrated that sensitive and chemically specific NMR spectra can be recorded in the absence of a magnetic field using hydrogenative parahydrogen induced polarization (PHIP) (1-3) and detection with an optical atomic magnetometer. Here, we show that non-hydrogenative parahydrogen-induced polarization (4-6) (NH-PHIP) can also dramatically enhance the sensitivity of zero-field NMR. We demonstrate the detection of pyridine, at concentrations as low as 6 mM in a sample volume of 250 μL, with sufficient sensitivity to resolve all identifying spectral features, as supported by numerical simulations.

Relieving bottlenecks in RNA drug discovery for retinal diseases.

The development of efficacious and safe post transcriptional gene silencing () agents is a challenging scientific endeavor that embraces “biocomplexity” at many levels. The target mRNA exhibits a level of structural complexity that profoundly limits annealing of PTGS agents. PTGS agents are macromolecular RNAs that must be designed to fold into catalytically active structures able to cleave the target mRNA.

A first-principles description of proton-driven spin diffusion.

Herein we design a reduced Liouville space for the simulation of proton-driven spin diffusion. Using this approach, the experimentally observed carbon-13 polarisation transfer in a powder sample undergoing magic-angle spinning is quantitatively described, directly from crystal geometry and without any adjustable parameters.

Nanoscale torsional resonator for polarization and spectroscopy of nuclear spins.

We propose a torsional resonator that couples to the transverse spin dipole of an attached sample. The absence of relative motion eliminates a source of friction that would otherwise hinder nanoscale implementation. Enhanced spontaneous emission induced by the resonator relaxes the longitudinal spin dipole at a rate of ∼1  s⁻¹ in the low-temperature limit.

Numerical simulation of free evolution in solid-state nuclear magnetic resonance using low-order correlations in Liouville space.

The design of simulations of free evolution in dipolar-coupled nuclear-spin systems using low-order correlations in Liouville space (LCL) is discussed, and a computational scheme relying on the Suzuki-Trotter algorithm and involving minimal memory requirements is described. The unusual nature of the approximation introduced by Liouville-space reduction in a spinning solid is highlighted by considering the accuracy of LCL simulations at different spinning frequencies, the quasiequilibria achieved by spin systems in LCL simulations, and the growth of high-order coherences in the exact dynamics. In particular, it is shown that accurate LCL simulations of proton spin diffusion occur in a regime where the reduced space excludes the coherences that make the dominant contribution to ∥σ∥(2), the norm-squared of the density matrix.

Ab initio simulation of proton spin diffusion.

The many-body nature of the ubiquitous spin diffusion phenomenon makes it difficult to predict accurately from first principles. We show how the use of reduced Liouville spaces makes it possible to reproduce experimental proton spin diffusion measurements directly from crystalline geometry for powdered solids under magic-angle spinning.

A high-throughput biophotonics instrument to screen for novel ocular photosensitizing therapeutic agents.

Purpose: High-throughput techniques are needed to identify and optimize novel photodynamic therapy (PDT) agents with greater efficacy and to lower toxicity. Novel agents with the capacity to completely ablate pathologic angiogenesis could be of substantial utility in diseases such as wet age-related macular degeneration (AMD).

Methods: An instrument and approach was developed based on light-emitting diode (LED) technology for high-throughput screening (HTS) of libraries of potential chemical and biological photosensitizing agents.

Bottlenecks in development of retinal therapeutic post-transcriptional gene silencing agents.

Development of post-transcriptional gene silencing (PTGS) agents for therapeutic purposes is an immense challenge in modern biology. Established technologies used to knockdown a specific target RNA and its cognate protein: antisense, ribozyme, RNAi, all conditionally depend upon an initial, critical annealing event of the PTGS ligand to a target RNA. In this review we address the nature of the bottlenecks, emphasizing the biocomplexity of target RNA structure, that currently limit PTGS therapeutic development.