NMR and UV studies of 3'-S-phosphorothiolate modified DNA in a DNA : RNA hybrid dodecamer duplex; implications for antisense drug design

High-resolution NMR spectroscopy has been used to establish the conformational consequences of the introduction of a single 3[prime or minute]-S-phosphorothiolate link in the DNA strand of a DNA : RNA hybrid. These systems are of interest as potential antisense therapeutic agents. Previous studies on similarly modified dinucleotides have shown that the conformation of the sugar to which the sulfur is attached shifts to the north (C(3[prime or minute])-endo/C(2[prime or minute])-exo). Comparisons made between NOESY cross-peak intensities, and coupling constants from PE-COSY spectra, for both non-modified and modified duplexes confirm that this conformational shift is also present in the double helical oligonucleotide system. In addition it is noted that in both the dinucleotides and the modified duplex, the conformation of the sugar ring 3[prime or minute] to the site of modification is also shifted to the north. That this pattern is observed in the small monomeric system as well as the larger double helix is suggestive of some pre-ordering of the sequences. The conclusion is supported by consideration of the (1)H chemical shifts of the heterocyclic bases near the site of the modification. The enhanced stability that these conformational changes should bring was confirmed by UV thermal melting studies. Subsequently a series of singly and doubly 3[prime or minute]-S-phosphorothiolate-modified duplexes were investigated by UV. The results are indicative of an additive effect of the modification with thermodynamic benefit being derived from alternate spacing of two modified linkers.     

Phase sensitive demodulation in multiphoton microscopy.

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.     

Experimental studies on the applicability of the Kelvin equation to highly curved concave menisci

The thermodynamic properties of liquids trapped in microscopic pores are described in theory by the Kelvin equation, which relates the equilibrium meniscus curvature to the relative vapor pressure. We report here two series of experiments designed to test the validity of the Kelvin equation by direct measurement of the mean radius of curvature of the surface of cyclohexane condensed between crossed mica cylinders. In one series of experiments, the relative vapor pressure of the volatile cyclohexane was controlled by mixing it with a relatively involatile solute (n-dodecane or n-hexadecane). We found that the mean radius of curvature rapidly reached that predicted by the Kelvin equation at each relative vapor pressure of the volatile liquid, but that there was also a slow, but continuous, accumulation of the “involatile” solute at the point of condensation as the system approached true equilibrium. Such accumulation of very low vapor pressure materials may be one factor responsible for the discordant results reported by earlier workers. We find that the process of impurity buildup is complex, and suggest that studies of real porous systems may be affected by accumulation of “involatile” impurities through the vapor phase and by surface diffusion. The other series of experiments was designed to eliminate the impurity problem by maintaining the vapor pressure by temperature control of the pure liquid. The results from this series of experiments were not time dependent, and no evidence of contamination was found. The measured radii were within ±6% of those predicted by the Kelvin equation, for radii in the range 4–20 nm. We conclude that the thermodynamic basis of the Kelvin equation is valid in principle for menisci with radii as low as 4 nm.