Mary E. Hawkins

Dideoxyinosine in children with symptomatic human immunodeficiency virus infection.

Abstract Background. 2′,3′-Dideoxyinosine (ddI) is a dideoxynucleoside with potent activity in vitro against the human immunodeficiency virus (HIV). In initial clinical trials in adults, ddI showed evidence of antiretroviral activity with little hematologic toxicity. Methods. We conducted a phase I–II study in 43 children with symptomatic (CDC class P-2) HIV infection. Of these children, 16 (median age, 10 years) had previously received zidovudine, and 27 (median age, 2.6 years) had not. ddI was administered orally in three divided doses totalling 60, 120, 180, 360, or 540 mg per square meter of body-surface area per day for 24 weeks. Eight of the 43 patients did not complete 24 weeks of ddI: 6 died, 1 was withdrawn because of progressive disease, and the other because of toxicity. Results. After oral administration, ddI was rapidly absorbed, although its bioavailability varied greatly among patients. Pancreatitis developed in two children, one receiving ddI at each of the two highest doses. The median CD...

Fluorescent Nucleoside Analogues as DNA Probes

The availability of a large selection of fluorescent probes has revolutionized our approaches to studying biochemical reactions and interactions. These probes have become so commonly used and important to many areas of research that it is hard to imagine working without them. This chapter will focus on a more recently developed subclass of DNA probes, nucleoside analogues, which are different from conventional probes in ways that provide opportunities for much more direct approaches in DNA research. In particular, the focus will be on these analogues used as probes incorporated into DNA. The source of their behavioral differences may not be apparent at first glance. An understanding of the differences between the carbon linker type fluorophores and the nucleoside analogues, and their physical relationships within the DNA will make this clear. The value and usefulness of the rich variety of fluorescent probes currently available is indisputable. They are, however, poorly suited for the type of experiments defined in this chapter. Because the majority of fluorophores are larger than and structurally dissimilar to purines or pyrimidines, they must be placed on a linker arm at some distance from the site of interest on the DNA. While this placement allows them to be used without perturbing the system, in many cases it removes them from the subtle interactions which are often the target of such investigations. The nucleoside analogues discussed within this chapter include only those probes that incorporate into an oligonucleotide through a deoxyribose linkage, are formulated as phosphoramidites to allow site-selective insertion, and participate in base-stacking (and sometimes base-pairing) interactions within the DNA. It is the base-stacking and base-pairing features that allow us to monitor subtle changes in DNA structure, binding, and composition. Probes that fit these require-

Pteridine Nucleosides—New Versatile Building Blocks in Oligonucleotide Synthesis

Abstract Chemical syntheses of 1-(2-deoxy-β-D-ribofuranosyl)lumazines and isopterins as well as 8-(2-deoxy-β-D-ribofuranosyl)-4-amino-7(8H)pteridones and -isoxanthopterins have been developed to make the structural analogs of the naturally occurring 2′-deoxyribonucleosides in the pteridine series available. The corresponding phosphoramidites have been used in machine-aided solid-support syntheses leading to new types of fluorescence labeled oligonucleotides. The effects of the various fluorophors on duplex formation and as labels for enzyme reactions is demonstrated.

Probing Variations In The Structural Environment Of A DNA Sequence Using Fluorescence Properties Of The Pteridine Analog Probes, 3MI and 6MI

We explored two different microenvironments in the sequence; 5′-actaGagatccctcagacccttttagtcagtGtgga -3′ in single and duplex form using two similar nucleoside analogs. 3MI and 6MI were each investigated in two different environments, one flanked by thymines (PTRT) and the other, by adenines (PTRA)(shown by Gs noted above). Each site is equidistant from a terminus. The probes differ only by the position of a methyl group in either the 3- (3MI) position or the 6- (6MI) position. Both time-resolved anisotropies and lifetimes of the probes depend upon local electrostatics which are impacted by duplex formation. 3MI shows less response to structural change as compared to 6MI. Integrals of lifetime curves compared with quantum yields of each sample reveal that each displays a “dark” component which we are unable to detect with TCSPC (e.g.,tau<70ps). For 6MI in the A environment this QSSQ “quasi static quenching” eliminates approximately half the molecules, whether in SS or DS form. 6MI in the T environment displays an unexpected increase in the quantum yield upon duplex formation (0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66% to 33%. Escape from the dark state is accompanied by doubling of steady state anisotropy of 6MI in PTRT in the duplex. Only 6MI in the T duplex displays a rotational correlation time over 7 ns. The DS A environment fails to constrain local motion and QSSQ remains the same as in SS; in contrast, the flanking T duplex environment restricts local motion and halves the QSSQ.