Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

A Cable-actuated Prosthetic Emulator for Transradial Amputees.

Upper limb prosthesis has a high abandonment rate due to the low function and heavyweight. These two factors are coupled because higher function leads to additional motors, batteries, and other electronics which makes the device heavier. Robotic emulators have been used for lower limb studies to decouple the device weight and high functionality in order to explore human-centered designs and controllers featuring off-board motors. In this study, we designed a prosthetic emulator for transradial (below elbow) prosthesis to identify the optimal design and control of the user. The device only weighs half of the physiological arm which features two active wrist movements with active power grasping. The detailed design of the prosthetic arm and the performance of the system is presented in this study. We envision this emulator can be used as a test-bed to identify the desired specification of transradial prosthesis, human-robot interaction, and human-in-the-loop control.

Design, Development, and Control of a Fabric-Based Soft Ankle Module to Mimic Human Ankle Stiffness.

This paper investigates the design of a robotic fabric-based, soft ankle module capable of generating 50% of the human ankle stiffness, in plantarflexion and dorsiflexion for walking. Kinematics, dynamics, and anatomy of the human ankle joint are studied to set the functional requirements of the module. The design of the compliant and lightweight soft ankle module uses fabric-based inflatable actuator arrays for actuation. Models for the human ankle stiffness, as well as a data-driven model of soft ankle module is presented. A high-level stiffness controller utilizing the human ankle and soft ankle model with a low-level pressure controller is implemented. We demonstrate the ability to closely follow the ankle stiffness trajectory using soft ankle module.