With controlled aeration, pH, and dissolved oxygen, these reaction vessels permit growth of our E. coli strains to optical densities exceeding 30, which provides us with ~300 gm of biomass from a 10-liter fermentation in ~12 hours. Working at this scale also requires an efficient procedure for rapid harvesting of the biomass. To achieve this, a Hereaus Contifuge T7 Stratos continuous-action centrifuge is connected directly to the biofermentor. This approach allows us to harvest the 10-liter reaction volume in less than 45 minutes. In addition to large-scale RT production for biophysical analysis, several retroviral and retrotransposon enzymes are currently under investigation, where purification in the 1-5 mg range is sufficient. For these needs, the laboratory is equipped with three independently controlled Innova platform shakers (right), each of which is capable of holding up to 6 2-liter culture flasks.

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Large-Scale Protein Purification While biofermentation provides us with the necessary quantities of biomass, this must be complemented with efficient and rapid protein purification methodologies. A dedicated cold laboratory contains HPLC/FPLC, FPLC, and low-pressure chromatographic equipment to meet these needs (left). For each instrument, a selection of affinity, ion exchange, and gel permeation matrices are available in both analytical and preparative scales. However, as the quantity of biomass increases, the use of chromatographic techniques at early purification steps becomes impractical and necessitates the application of batch strategies, which are likewise conducted in the cold laboratory. A second workbench in the cold laboratory is reserved for low-temperature electrophoresis when fractionating nucleic acid duplexes by nondenaturing polyacrylamide gel electrophoresis.

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Oligonucleotide and Peptide Separation Several projects recently initiated in the laboratory involve purification of nucleic acids and peptides by HPLC. Examples of this include the synthesis of oligonucleotides containing proteolytic, nucleolytic, and photoactivable bioconjugates. In addition, modified bases such asinosine and 2-aminopurine are being incorporated into nucleic acids to probe the structure of retroviral reverse transcription complexes. Finally, in conjunction with mass spectrometry, proteolytic fragments of p66/p51 HIV-1 RT are separated by HPLC for analysis by microsequencing. For such projects, the laboratory makes use of a Beckman/Coulter System Gold HPLC equipped with a diode array, fluorescence detector, and column oven (right).

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Molecular Modeling Our structure/function studies with HIV-1 RT have been aided by the availability of several high-resolution crystal structures of the p66/p51 enzyme. These include (a) the unliganded enzyme, (b) co-crystals with several nonnucleoside-based inhibitors, (c) a binary complex containing duplex DNA, (d) a ternary complex of enzyme, DNA, and an incoming deoxynucleoside triphosphate, and (e) an RNA-DNA hybrid encompassing the polypurine tract. Molecular modeling thus plays a pivotal role in interfacing our biochemical studies on mutant enzymes with the consequences for the structure of either the p66 or p51 subunit. In addition, using a combination of biochemistry and molecular modeling, we are investigating several small-molecule inhibitors of RNase H function that have been identified recently. Researchers in the RT Biochemistry Section receive instruction on molecular modeling using a Silicon Graphics Octane 2 workstation with staff of the Advanced Biomedical Computing Center (left).

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Protein Footprinting via Mass Spectrometry A variety of chemical and enzymatic probes are available to study changes in nucleic acid conformation upon binding of ligand. However, solution methodologies providing high-resolution information on the protein component of this complex are considerably less developed and have traditionally relied on tagging the protein at its N- or C-terminus with an epitope or phosphorylation site. Mass spectroscopy is rapidly emerging as a simple yet highly precise approach that can be applied to this problem. Nucleoprotein complexes are subjected to hydrolysis by one of several highly specific endoproteinases, after which the products are resolved by MALDI-TOF. The high degree of accuracy of the mass spectrometer thereafter allows the assignment of peaks to peptides of the original protein. In collaboration with the NCI Laboratory of Medicinal Chemistry, researchers of the RT Biochemistry Section make use of a Kratos Kopmact SEQ mass spectrometer equipped with a 1.7-meter flight tube for enhanced resolution (right). Peptide mixtures analyzed by the mass spectrometer can also be verified by their separation via HPLC and peptide sequencing. [Top of page] Laser Crosslinking Bioconjugation (i.e., site-specific tethering of two molecules to generate a novel complex displaying the combined properties of its individual components) is a powerful complement to high-resolution crystallographic and spectroscopic methods in providing structural information on protein-nucleic acid complexes. Projects currently underway in the laboratory involve attachment of artificial nucleases and proteases to the protein and nucleic acid components of nucleoprotein complexes. In addition, site-specific attachment of photocrosslinking agents provides high-resolution structural information on the interaction of RT with its conformationally distinct substrates. In order to probe the interaction of RT with template and primer nucleotides in real time (i.e., in the millisecond range). The laboratory makes use of a New Wave "Tempest" pulse laser in combination with a Kin-Tek rapid-quench apparatus (left). Laser crosslinking is also in use to evaluate the binding site of small-molecule RNase H antagonists. [Top of page] In Vivo Studies with HIV and FIV Although projects in the laboratory have to this stage been primarily of a biochemical nature, studies involving culture of HIV and feline immunodeficiency virus (FIV) have recently been initiated. These include control of reverse transcription during initiation of (-) strand DNA synthesis in FIV and evaluation of central termination in HIV. In vitro experimentation suggests that a novel interaction of tRNALys,3 and the FIV genome controls initiation, and efforts are underway to study this long-range intermolecular interaction through alterations of the FIV genome. A second project involves the specificity of cPPT/CTS elements, which are proposed to play a structural role at a late step in the HIV reverse transcription cycle. Similarity between these elements of HIV and equine infectious anemia virus is under investigation. For such studies, the laboratory makes use of cell culture facilities (right) in the laboratory of Dr. Vineet KewalRamani. [Top of page] Centrifugal Filtration During large-scale protein purification, exchanging buffers and desalting macromolecular solutions via dialysis become both impractical and time consuming. Centrifugal filtration, using filtration devices with a variety of membrane cut-offs, permits the rapid concentration of solutions in volumes ranging from 100 ml (using filters inserted into Eppendorf microcentrifuge tubes) to 1000 ml, where 200-ml devices are used in combination with high-speed tabletop centrifuges (left). Low adsorbtion of the cellulose membrane and the components of the filtration device combine to give recoveries in excess of 95% with a processing time of 10-60 minutes. [Top of page] Specialized Oligonucleotide Synthesis Introducing modified nucleosides into synthetic DNA and RNA oligonucleotides by phosphoramidite chemistry allows us to examine in considerably more detail the interaction of RT with the structurally distinct nucleic acid duplexes encountered during replication. These substrates include duplex DNA, duplex RNA, and RNA/DNA hybrids. Our recent studies have involved introducing non-hydrogen-bonding pyrimidine isosteres (or shape mimics) into DNA to evaluate how the flexibility of DNA/RNA hybrids influences their recognition by the RTs of HIV-1 (Rausch et al., PNAS, 2003) and the Saccharomyces cerevisiae LTR-retrotransposon Ty3 (Lener et al., JBC, 2003). Another study has taken advantage of the unique fluorescence properties of the cytidine analog, pyrrolo-dC, whose emission spectrum is considerably removed from that of tryptophan, to explore hydrogen-bonding patterns in RNA/DNA hybrids (Dash et al., NAR, 2004, in press). Lastly, the unique properties of certain nucleoside analogs can be exploited to study the conformation of RNA/DNA hybrids by NMR spectroscopy. This combination of biophysical and biochemical studies clearly requires multi-milligram quantities of both DNA and RNA oligonucleotides containing modified bases. Synthesis of specialized oligonucleotides is conducted by individual researchers of the RT Biochemistry Section, each of whom has received training from Dr. Yi-Brunozzi (right), a specialist in RNA synthesis. [Top of page] Nonradioactive Electrophoretic Methods: Capillary Electrophoresis Conventionally, analyzing the enzymatic activities of RT has required 5' or 3' end-labeling of DNA or RNA oligonucleotides with [32P], resolving reaction products by high-resolution denaturing gel electrophoresis, and their quantitation by densitometry or phosphorimaging. Substituting radioactive procedures with colorimetric or fluorescent assays, while at the same time allowing the same sample throughput, would clearly be advantageous. Using phosphoramidite chemistry, fluorescent tags such as fluorescein can be introduced within or at the termini of both DNA and RNA. Following RNase H-mediated hydrolysis or polymerase-mediated DNA synthesis, the reaction products can be separated by capillary electrophoresis and quantified by laser-induced fluorescence. The instrument illustrated (left) will resolve RNase H-derived hydrolysis products in 5-7 minutes, and at the some time provides direct quantitation and single-nucleotide separation. In addition, its 96-well platform allows multiple samples to be loaded and analyzed overnight. In collaboration with researchers of the NCI Molecular Targets Development Program and SAIC Analytical Chemistry Laboratory, capillary electrophoresis plays a central role in high-throughput screening of inhibitors of HIV-1 and human RNase H. Additional projects of the RT Biochemistry Section involve adapting the technology for both steady-state and pre-steady-state analysis of the RTs of HIV-1 and Ty3. When direct visualization of reaction products is not necessary (i.e., when simply the extent of RNase H-mediated hydrolysis must be measured), a variation of the fluorescence approach can again be used. In this case, the RNA and DNA components of an RNA/DNA hybrid contain a fluorescence donor and quencher, respectively, the proximity of which results in fluorescence quenching. Following RNase H-mediated hydrolysis, the fluorescence donor is relieved of the quenching environment, resulting in a simple, sensitive, and quantifiable "off/on" RNase H assay. Using a fluorescence plate reader, samples can once more be evaluated in a 96-well format (right). The two RNase H assays described briefly here, in conjunction to DNA polymerase assays under development, represent considerable savings in both time and cost, as well as reducing the environmental burden associated with storage and disposal of radioactive waste.

Last modified: 22 December 2008