Tobias Restle

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes.

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.

Factors contributing to the inhibition of HIV reverse transcriptase by chain‐terminating nucleotides in vitro and in vivo

Arguments are presented leading to the conclusion that two major factors contribute to the potency of inhibition of DNA‐polymerase activity by chain‐terminating nucleotides. The relative significance of these factors varies with the reaction conditions, particularly with the length of the template and the concentration ratio of enzyme (reverse transcriptase or other DNA polymerase) to primer. It is concluded that potent inhibition of HIV‐reverse transcriptase activity under typical in vitro and in vivo conditions arises from different features of the interaction of chain terminators with the enzyme. A new method of testing for the parameter important under in vivo conditions is suggested.

RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV‐1 and HIV‐2 reverse, transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes

Characterization of the dimerization process of HIV-1 reverse transcriptase heterodimer using intrinsic protein fluorescence

Intrinsic protein fluorescence has been used to study dimerization of the HIV‐1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two‐state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (± 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100‐fold acceleration of the association by the divalent ion was observed.

Selected Strategies for the Delivery of siRNA In Vitro and In Vivo

RNA-based therapeutic strategies are considered as a highly promising alternative to conventional drug development. Among the different classes of oligonucleotide-derived prospective drugs, small interfering RNAs (siRNAs) are of particular interest. However, cellular uptake and subsequent intracellular trafficking to the effector complex (RNA-induced silencing complex; RISC) represent major technical hurdles for the efficacy of these macromolecular drugs. Thus, the development of appropriate delivery systems is an essential requirement to turn these molecules into medicine. In this review, we will focus on two particular auspicious aspects in this context, the phosphorothioate-stimulated uptake of naked siRNA and the use of cell-penetrating peptides as shuttles for a controlled cellular uptake. Moreover, we will present some of the most promising recent approaches for siRNA delivery in vivo, which may help to pave the road to drugs of the future.

Pre-steady-state kinetic characterization of the DinB homologue DNA polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is ∼10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s-1 for dTTP, dATP, dGTP, and dCTP (at 25 °C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (Kd ∼600 μm), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an “induced fit” mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Studies on the Interaction between HIV-1 Reverse Transcriptase and Templateoligo α-Thymidylate Duplexes

Abstract Two methods have been used to determine the affinity to HIV-1 reverse transcriptase of duplexes between α-dTn, and an RNA template. In one of these, competition between the duplexes and a fluorescently labeled temp1ateprimer complex was used to provide equilibrium affinity data. In the second method, the duplexes were used as competitors for poly Aoligo dT in a standard reverse transcriptase assay. Both approaches indicated that substitution of β-nucleoside units by their α-analogs led to a 2–10 fold lowering of the affinity to HIV-1 reverse transcriptase.