Dynamic behavior of DNA base pairs containing 8-oxoguanine

The process by which DNA repair enzymes recognize and selectively excise damaged bases in duplex DNA is fundamental to our mechanistic understanding of these critical biological reactions. 8-Oxoguanine (8-oxoG) is the most common form of oxidative DNA damage; unrepaired, this lesion generates a G:C-->T:A mutation. Central to the recognition and repair of DNA damage is base extrusion, a process in which the damaged base lesion or, in some cases, its partner disengages from the helix and is bound to the enzyme's active site where base excision takes place. The conformation adopted by 8-oxoG in duplex DNA is affected by the base positioned opposite this lesion; conformational changes may also take place when the damaged base binds to its cognate repair enzyme. We performed unrestrained molecular dynamics simulations for several 13-mer DNA duplexes. Oligomers containing G:C and 8oxoG:C pairs adopted Watson-Crick geometries in stable B-form duplexes; 8oxoG showed increased local and global flexibility and a reduced barrier to base extrusion. Duplexes containing the G:A mismatch showed much larger structural fluctuations and failed to adopt a well-defined structure. For the 8oxoG:A mismatch that is recognized by the DNA glycosylase MutY, the damaged nucleoside underwent spontaneous and reproducible anti-->syn transitions. The syn conformation is thermodynamically preferred. Steric hindrance and unfavorable electrostatics associated with the 8oxoG O8 atom in the anti conformation were the major driving forces for this transition. Transition events follow two qualitatively different pathways. The overall anti-->syn transition rate and relative probability of the two transition paths were dependent on local sequence context. These simulations indicate that both the dynamic and equilibrium behavior of the duplex change as a result of oxidation; these differences may provide valuable new insight into the selective action of enzymes on damaged DNA.     

Grating-enhanced through-wafer optical microprobe for microelectromechanical system high-resolution optical position feedback

We present modeling and experimental results from the use of a 1310-nm-wavelength through-wafer optical microprobe in conjunction with a microstructure grating to monitor the motion of a lateral comb resonator stage. The optical signal that results from shuttle interaction with the microprobe beam exhibits a peak-to-valley dynamic range that corresponds to 2-microm microstructure displacement, facilitating submicrometer positional resolution on digitization. This signal was used to achieve microstructure positional feedback and effective microsystem model parameter extraction, which are essential for structure control and model-based fault detection.     

Elementary single turn solenoids used as the transmitter and receiver in magnetic resonance imaging

A single turn solenoid, also called a loop-gap resonator, is a device that is efficient for radio frequency spectroscopy on relatively large samples. Thus, the device provides an effective means for magnetic imaging where the single turn solenoid may serve both as the transmitter and receiver coil. The device is readily constructed and provides very efficient use of radio frequency (RF) power for imaging extremities such as breasts, arms, feet, and hands. The resulting magnetic images are acquired in short times with good anatomical resolution and considerable reduction of the RF power delivered to the patient.     

A volume resolution phantom for MRI

Multisite quantitative magnetic resonance imaging (qMRI) of volume requires a small isotropic point spread-function (PSF) that is spatially, temporarily, and platform invariant. A phantom which will allow rapid assessment of this metric throughout the imaged volume without repositioning will assist certification of imaging sites for use in qMRI studies based on volume. This paper presents a phantom design for this purpose with a three-dimensional repeating pattern throughout its 800-cm³ volume. The image of the pattern from the phantom contains a series of positive signal points and lines which can be used to measure the PSF, gradient linearity, gradient orthogonality, and B₀ homogeneity at multiple locations throughout its volume. The phantom is readily constructed, can be filled with any nuclear magnetic resonance signal-bearing liquid, and the design is scalable to cover larger volumes.     

The open structure of a multi-drug-resistant HIV-1 protease is stabilized by crystal packing contacts

The introduction of HIV-1 protease (HIV-PR) inhibitors has led to a dramatic increase in patient survival; however, these gains are threatened by the emergence of multi-drug-resistant strains. Design of inhibitors that overcome resistance would be greatly facilitated by deeper insight into the mechanistic events associated with binding of substrates and inhibitors, as well as an understanding of the effects of resistance mutations on the structure and dynamic behavior of HIV-PR. We previously reported a series of simulations that provide a model for HIV-PR dynamics, with spontaneous conversions between the bound and unbound crystal forms upon addition or removal of an inhibitor. Importantly, the unbound protease transiently sampled a third fully open state that permits entry to the active site, unlike both crystallographic forms. Recently, a crystal structure of unbound HIV-PR was reported for the MDR 769 isolate (PDB: 1TW7); unlike all previous experimental structures, the binding pocket is open. It is suggested that drug resistance in this strain arises at least in part from the inability of inhibitors to induce closing. We carried out simulations of the MDR 769 HIV-PR mutant and observed that the reported structure is unstable in solution and rapidly adopts the semi-open conformation observed for the unbound wild-type protease in solution. Further analysis suggests that the wide-open structure observed for MDR 769 arises not from sequence variation, but instead is an artifact from crystal packing. Thus, despite being the first experimental structure to reveal flap opening sufficient for substrate access to the active site, this structure may not be directly relevant to studies of inhibitor entry or to the cause of HIV-PR drug resistance.     

Modular Synthesis of DOTA-Metal-Based PSMA-Targeted Imaging Agents for MRI and PET of Prostate Cancer

A practical, convergent synthesis of prostate-specific membrane antigen (PSMA) targeted imaging agents for MRI, PET, and SPECT of prostate cancer has been developed. In this approach, metals chelated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were placed on the side chains of lysine early in the synthesis to form imaging modules. These are coupled to targeting modules, in this case consisting of the PSMA-binding urea DCL, bonded to an activated linker. The modular approach to targeted molecular imaging agents (TMIAs) offers distinct advantages. By chelating the MRI contrast metal Gd early, it doubles as a protecting group for DOTA. Standard coupling and deprotection steps may be utilized to assemble the modules into peptides, and the need for tri-tert-butyl protection of DOTA requiring removal by strong acid is averted. This enables mild conjugation of the imaging module to a wide variety of targeting agents in the final step. It was further discovered that two labile metals, La³⁺ or Ce³⁺, can be used as placeholders in DOTA during the synthesis, then transmetalated in mild acid by Cu²⁺, Ga³⁺, In³⁺, and Y³⁺, metals used in PET/SPECT. This enables the efficient synthesis of nonradioactive analogues of targeted molecular imaging agents that may be transported or stored until needed. A simple and mild two-step transmetalation, involving de-metalation in dilute acid, followed by rapid chelation of the radioactive metal, may be conveniently performed later at the clinic to provide the TMIAs for PET or SPECT.     

Unilateral MRI using a rastered projection

Unilateral magnetic resonance techniques, where magnet and radio frequency (RF) coil are placed on one side of the sample, can provide valuable information about a sample which otherwise cannot be accommodated in conventional high spectral resolution magnetic resonance systems. A unilateral magnetic resonance imaging approach utilizing the stray field from a disc magnet and a butterfly geometry RF coil is described. The coil excites spins in a volume centered around an arc through the sample. Translating the RF coil relative to the magnet and recording the signal at each translational location creates a projection of the signal in a tomographic slice through the sample. Rotating the RF coil relative to the sample and repeating the translation creates projections through the sample at different angles. Backprojecting this information yields an image. A proof of concept device operating on this principle at 12.4 MHz was constructed and characterized. Projections through three phantoms are presented with a 1.2-4 cm field of view, thickness of 102 microm, and at a distance of 3mm from the RF coil and 14 mm from the magnet. The edge spread function (ESF) was measured resulting in a 4mm full width at half maximum (FWHM) line spread function (LSF) estimation using a Gaussian model. An example of one reconstructed image is presented.