Recent advances in the synthesis and structure determination of site specifically psoralen-modified DNA oligonucleotides.

We have developed novel methods for the preparation of multimicromole quantities of extremely pure, uniquely photoadducted psoralen-DNA cross-links, furan-side monoadducted DNA and pyrone-side monoadducts. Psoralen cross-linked and furan-side monoadducted DNA were produced by employing high intensity argon ion and krypton ion lasers as light sources. Pyrone-side monoadducts were prepared by base-catalyzed photoreversal of psoralen cross-links. The various psoralen-adducted DNA oligomers were efficiently purified by high performance liquid chromatography. These methods have permitted us to synthesize 4 mumol each of a self-complementary 8-mer d(GCGTACGC) 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furan-side monoadduct and HMT cross-link. Preliminary nuclear magnetic resonance (NMR) data on the HMT cross-linked 8-mer d(GCGTACGC) have been obtained which confirmed the presence of the diadducted psoralen at the unique 5'TpA3' site. NMR data obtained from the 8-mer furan-side monoadduct revealed that the psoralen molecule is intercalated into the DNA double helix. Preliminary crystals of 8-mer cross-linked DNA molecule have been grown. Conditions for the growth of X-ray diffraction-quality crystals and the further analysis of these crystals are now in progress.     

Quantification and adjustment of pixel-locking in particle image velocimetry

A quantification metric is provided to determine the degree to which a particle image velocimetry data set is pixel-locked. The metric is calculated by integrating the histogram equalization transfer function and normalizing by the worst-case scenario to return the percentage pixel-locked. When this metric is calculated for each position in the vector field, it is shown that pixel-locking is non-uniform across the field. Hence, pixel-locking adjustments should be made on a vector-by-vector basis rather than uniformly across a field, although the latter is the common practice. A methodology is provided to compensate for the effects of pixel-locking on a vector-by-vector basis. This includes applying a Gaussian filter directly to the images, processing the images with window deformation, ensuring the vector fields are in pixel displacements, applying histogram equalization calculated at each vector coordinate, and mapping the adjusted vector fields to physical space.     

THE MOLECULAR and STEREOCHEMICAL STRUCTURES OF PHOTOPRODUCTS GENERATED BY UV-IRRADIATION OF 4''-HYDROXYMETHYL-4,5'',8''- TRIMETHYLPSORALEN IN AQUEOUS SOLUTION

UV irradiation of 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in aqueous solution yields three major photoproducts. We have isolated and characterized (1) a cyclobutane-bridged dimer in which a cis-syn linkage occurs between the furan end of one HMT moiety and the pyrone end of the other; (2) a cyclobutane-bridged dimer wherein both HMT moieties are linked at their pyrone ends with probable cis-syn configuration; and (3) an isomer of HMT for which we have proposed a structure in which the furan and pyrone ring oxygens assume a para orientation via photoisomerization.     

Experimental estimation of fluctuating velocity and scalar gradients in turbulence

The effect of numerical differentiation is investigated in the context of evaluating fluctuating velocity and scalar quantities in turbulent flows. In particular, 2-point forward-difference and 3-, 5-, 7-, and 9-point centred-difference schemes are investigated. The spectral technique introduced by Wyngaard (in J Sci Instr 1(2):1105–1108, 1968) for homogeneous turbulence is used to quantify the effects of the schemes. Numerical differentiation is shown to attenuate gradient spectra over a range of wavenumbers. The spectral attenuation, which varies with the order of the scheme, results in a reduction in the measured mean-squared gradients. High-order schemes (e.g. 7- or 9-point) are shown to significantly decrease the attenuation at all wavenumbers and as a result produce more accurate gradients. Hot-wire measurements and direct numerical simulations of decaying homogeneous, isotropic turbulence are found to be in good agreement with the predictions of the analysis, which suggests that high-order schemes can be used to improve empirical gradient estimates. The shape of the probability density functions is also found to be sensitive to the choice of numerical differentiation scheme. The effect of numerical differentiation is also discussed with respect to particle image velocimetry (PIV) measurements of a nominally two-dimensional planar mixing layer. It is found that the relatively low signal-to-noise ratio inherent in typical PIV measurements necessitates the use of low-order schemes to prevent excessive noise amplification, which increases with the order of the scheme. The results of the present work demonstrate that high-order numerical differentiation schemes can be employed to more accurately resolve gradients measured at a given resolution provided the measurements have an adequate signal-to-noise ratio.     

EFFICIENT FORMATION OF A CROSSLINKABLE HMT MONOADDUCT AT THE Kpn I RECOGNITION SITE

Abstract The double-stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten-fold more susceptible to HMT modification than the Bam HI site. Selective reaction with the Kpn I site is attributed to preferential intercalation of HMT into the TpA sequence. The availability of crosslinked Kpn I linker has allowed us to characterize the kinetics of photoreversal. Upon irradiation at 254 nm, the diadduct reverses at a faster rate than the monoadduct, leading to an accumulation of the latter. The resultant monoadduct reforms crosslink rapidly upon irradiation at 365 nm. DNA oligomers like the Kpn I linker, which can be modified by the above protocol to contain a crosslinkable HMT monoadduct, could he exploited as photoreactive sequence-specific probes.     

CHARACTERIZATION OF THE PHOTOREACTION BETWEEN DNA AND AMINOMETHYL-TRIMETHYLPSORALEN USING ABSORPTION AND FLUORESCENCE SPECTROSCOPY

–The use of absorption and fluorescence spectroscopy for following the progress of the photo-reaction between DNA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been investigated. Absorption at long wavelengths and fluorescence both decline upon intercalation of AMT into the DNA helix. The loss of fluorescence from AMT and the accompanying appearance of monoadduct fluorescence upon irradiation by UV light can be easily followed by using the excitation beam of a spectro-fluorometer as the source of irradiation and monitoring the changing emission spectrum. Where cross-link formation is possible, the subsequent decline of monoadduct fluorescence is seen as well. This suggests that the 4,5-tnonoadduct is a precursor of cross-links. Both monoaddition and cross-linking are more rapid with poly d(A·T) than with calf thymus DNA or poly d(A·T). Excitation spectra can be helpful in resolving the levels of AMT and 4',5'-monoadduct when both are contributing to the emission spectrum. Some changes are observed in the emission spectrum of AMT–poly d(A·T) monoadducts after prolonged irradiation which indicate further photoreaction.