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Lab Tour of the RT Biochemistry Section

Le Grice Lab photo of research bay

Custom redesigned in 1999, the RT Biochemistry Section comprises approximately 2500 square feet of laboratory space on the 3rd floor of Building 535. The laboratory is divided into four units, namely (a) the main laboratory, which holds eight researchers, (b) a smaller laboratory, which accommodates three researchers and houses a variety of instrumentation Le Grice Lab figure, structure of RTfor nucleic acid and peptide synthesis and (c) a dedicated cold room for large-scale protein purification and low-temperature nondenaturing gel electrophoresis, and (d) a fermentation facility, comprising a biofermentor and continuous-action centrifuge. Each research bay contains ample storage space and undercounter +4oC and -20oC refrigerators, and researchers are provided with writing space, a laptop computer, and a shared laser printer. Most electrophoretic procedures in the laboratory have replaced radioisotopes with fluorescently labeled nucleic acids, and gels are scanned on a Typhoon Trio imager.

Le Grice Lab photo of Dr. Marion Bona training high school studentsThe research component of the laboratory is complemented by courses/meetings that provide different approaches for preparing, presenting, and discussing scientific data. The biweekly RT Biochemistry Section lab meetings, biweekly work-in-progress presentations of all HIV DRP fellows, and biweekly NCI-Frederick Postdoc Seminar Series provide opportunities to both present data and discuss projects of other fellows. In order to gain experience as independent scientists during their training at NCI, fellows are encouraged to attend the NIH Grants 101 course to familiarize themselves with the grant application and review process. This course is a stepping-stone toward applying for long-term independent career grants such as K99/R00. Outreach activities include hosting students from nearby high schools and on-site demonstrations of molecular biology protocols. Individual projects and related equipment of the laboratory are described below.


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Biofermentation

Le Grice Lab biofermentation figure, 1 of 2 Projects currently underway in the RT Biochemistry Section have required the preparation of HIV-1 RT in amounts varying from a few nanograms for evaluation of enzymatic activity, to several hundred milligrams for X-ray crystallography, single-molecule spectroscopy, and isothermal titration calorimetry. Moreover, enzymes such as T7 RNA polymerase, Taq DNA polymerase, and T4 RNA ligase are required in substantial quantities for routine manipulations in several projects. Large-scale biofermentation facilities thus serve a central role in each research theme currently under investigation. In order to meet these demands, the RT Biochemistry Section has invested in a dedicated biofermentation facility. The figure on the right illustrates a New Brunswick Bioflo 4500 5-20 liter, self-sterilizing, computer-controlled fermentor/ bioreactor operated by Dr. Marion Bona.

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 Le Grice Lab biofermentation figure, 2 of 2 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 (HIV, XMRV, FIV) and retrotransposon (Ty3) 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 (left), each of which is capable of holding up to six 2-liter culture flasks.



Large-Scale Protein Purification

Le Grice Lab large-scale protein purification figure, 1 of 2 While biofermentation rapidly and efficiently provides us with the necessary quantities of biomass, this must be complemented with rapid, large-scale protein purification methodologies. A dedicated cold laboratory contains three HPLC instruments. 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 Le Grice Lab large-scale protein purification figure, 2 of 2cold laboratory is reserved for low-temperature electrophoresis when fractionating nucleic acid duplexes by nondenaturing polyacrylamide gel electrophoresis. Several structure/function studies have benefitted from large-scale protein purification. These include (a) crystallization of HIV-1 RT in the presence of a nonnucleoside RT inhibitor and a non-PPT RNA/DNA hybrid, (b) a revised structure for the isolated RNase H domain of XMRV RT, and (c) a novel structure of HIV-1 RT containing a nonnucleoside RT inhibitor, an allosteric R Nase H inhibitor, and a DNA/RNA hybrid.

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Molecular Modeling

Our structure/function studies with both lentiviral (HIV-1) and gamma-retroviral retroviral (XMRV) RTs have been augmented by the availability of several high- resolution crystal structures of these enzymes in their unliganded and nucleic acid-bound state state, as well as cocrystals Le Grice Lab molecular modeling figure, 1 of 2containing inhibitors of both DNA polymerase and RNase H activity. More recently, we have been successful in obtaining multiple cocrystals of HIV-1 RT in the presence of a non-PPT-containing RNA/DNA hybrid, showing major structural changes in both the p66 and p51 subunit following nucleic acid binding. New interactions between structural elements at the p51 C-terminus and their potential role in drug resistance are under evaluation by site-directed mutagenesis. Related projects have resulted in a high-resolution structure for RT of the gammaretrovirus XMRV. In both instances, molecular modeling plays a pivotal role in interfacing our biochemical and biophysical studies. Additional projects of the RT Biochemistry Section are investigating the Le Grice Lab molecular modeling figure, 2 of 2tertiary structure of cis-acting elements of retroviral and LTR-retrotransposon genomes that mediate RNA transport (HIV-1 RRE) and genome packaging (HIV-1 RRE, Ty1 5'-3' pseudoknot). As a complement to chemoenzymatic probing by SHAPE, the recently published program RNA Composer allows automated prediction of RNA 3D structures from a user-defined secondary structure and provides large RNA 3D structures of high quality. As with our crystallography projects, the tertiary structures predicted by this program provide a platform for site-directed mutagenesis, genetics, and through-space RNA cleavage strategies.



Peptide Synthesis and Metallotherapeutics

Le Grice Lab peptide synthesis and metallotherapeutics figure, 1 of 2Located at the N-terminus of several proteins, N-terminal Cu2+/Ni2+-binding motifs (ATCUNs) are proposed to bind and transport transition metals in blood. However, the avidity with which they bind Cu2+ and Ni2+ (KD ~10-15 M) suggests additional biological roles, a notion supported by the ability of Cu2+-peptide complexes to catalyze DNA and protein scission in the presence of ascorbate. ATCUNs have been implicated in antimicrobial activity of the salivary peptide Histatin 5, antitumor activity against Erlich ascites tumor cells, and inhibition of histone deacetylase. The insert below illustrates an ATCUN motif (-Gly-Gly-His-), comprising an alpha-amino nitrogen, two intervening peptide nitrogens, and the imidazole nitrogen of His at the third position. ATCUN activity likely reflects Cu2+/Ni2+-catalyzed release of reactive oxygen species and oxidative damage via Fenton chemistry, thus defining them as chemical "proteases" and Le Grice Lab peptide synthesis and metallotherapeutics figure, 2 of 2"nucleases" capable of inducing irreversible damage into their target biomolecule. Since superoxide anions are released from hemoglobin-bound O2 at a significant rate, and super-oxide dismutase converts superoxide to H2O2 and O2, ATCUNs also provide a novel means of generating "catalytic" metallotherapeutics capable of circulating through, and inactivating, their target population. The ability to append ATCUNs to peptides offers the additional opportunity to develop "molecular rulers" for studying protein:protein and protein:nucleic acid interactions.

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Oligonucleotide Synthesis

Le Grice Lab oligonucleotide synthesis figure, 1 of 2Incorporating 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. Recent studies in the laboratory have involved introducing non-hydrogen-bonding shape mimics, or pyrimidine isosteres (see inset), into DNA to evaluate how the flexibility of DNA/RNA hybrids influences their recognition by the RTs of HIV-1 and the S. cerevisiae LTR-retrotransposon Ty3. Another study has taken advantage of the fluorescence properties of the cytidine analog, pyrrolo-dC, whose emission spectrum is considerably removed from that of tryptophan, to explore hydrogen-bonding patterns inLe Grice Lab oligonucleotide synthesis figure, 2 of 2 RNA/DNA hybrids. 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. Finally, since RNA structure analysis via high-throughput SHAPE exploits multiplexing with fluorescently labeled DNA oligonucleotides, their in-house synthesis is both rapid and cost effective. Oligonucleotide synthesis is carried out by individual researchers of the RT Biochemistry Section, each of whom receives training from senior lab personnel.


Differential Scanning Calorimetry

Le Grice Lab differential scanning calorimetry figure, 1 of 2 Differential scanning fluorimetry (DSF, alternatively known as ThermoFluor) has emerged as a rapid, sensitive, and cost-effective screening strategy to identify low-molecular-weight ligands whose binding stabilizes or destabilizes their target biomolecule. The technology can easily be applied to study the interactions of ligands with drug-resistant forms of the target. The temperature at which the target protein unfolds is monitored via increased fluorescence of a dye (Sypro Orange) with affinity for hydrophobic regions of the protein. As the protein unfolds, the hydrophobic regions become increasingly exposed, which bind the dye, increasing its fluorescence (see inset). A straightforward fitting procedure allows calculation of the transition midpoint, or Tm; the difference in Tm in the presence and absence of a ligand is related to its binding affinity. A variety of ligands, including low-molecular-weight compounds, peptides, and nucleic acids, can be analyzed using this procedure. DSF is best performed using a conventional real-time PCR instrument. Solutions containing potential ligands are added from a storage plate to a solution of protein Le Grice Lab differential scanning calorimetry figure, 2 of 2and dye, distributed into wells of the PCR plate, and the fluorescence intensity is measured as the temperature is raised gradually. Results can be obtained in a single day. Furthermore, in contrast to isothermal titration calorimetry (ITC), which uses substantial amounts of protein, DSF can be performed on a few micrograms, and can be conveniently performed in a 96-well format. DSF was recently used to characterize a novel class of allosteric inhibitors of HIV integrase, or ALLINIs, which stabilize tetramer formation and interfere with catalysis. This approach has also been employed used to characterize allosteric inhibitors of HIV-1 RNase H activity. In contrast to RNase H active-site inhibitors, which increase the Tm by ~2oC, thienopyrimidinones decrease the Tm, in some cases by as much as 5oC.

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High-Throughput Screening

Le Grice Lab high-throughput screening figure, 1 of 2The RT Biochemistry Section is currently involved in high-throughput screening to identify inhibitors of two key HIV-1 enzymes, namely the RT-associated RNase H and integrase. Both projects share a common theme inasmuch as they are directed toward allosteric inhibition rather than directly binding to the active site of the enzyme. With regard to RNase H, thieno-pyrimidinones have been identified that bind at the interface between the p66 and p51 RT subunits and likely indirectly influence the geometry of the DNA/RNA hybrid in the RNase H active site. The success of high-throughput screening relies on developing simple, nonradioactive assays that easily lend themselves to automation. When direct visualization of reaction products is not necessary (i.e., when simply the extent of RNase H-mediated hydrolysis must be measured), the RNA and DNA components of an RNA/DNA hybrid can be synthesized to contain a fluorescence donor and quencher, respectively, the proximity of which results in fluorescence quenching. Following RNase H-mediated hydrolysis, the fluorescence quencher is removed Le Grice Lab high-throughput screening figure, 2 of 2and the donor is thereby freed from the quenching environment, resulting in a simple, sensitive, and quantifiable "off/on" RNase H assay. Using a plate reader such as the Tecan Infinite M1000Pro illustrated here, samples are evaluated in a 96-well format, representing considerable savings in both time and cost, as well as reducing the environmental burden associated with storage and disposal of radioactive waste. The Tecan Infinite M1000Pro is also equipped with time-resolved FRET, fluorescence polarization, glow luminescence, and flash luminescence capabilities.



Capillary Electrophoresis

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) is a facile approach to probe the structure of RNA at single-nucleotide resolution. This methodology employs an electrophilic reagent that reacts selectively with the 2-hydroxyl Le Grice Lab capillary electrophoresis figure, 1 of 2ribose group to create covalent 2-O-ribose adducts. SHAPE interrogates local nucleotide dynamics, as single-stranded nucleotides are more prone to adopt conformations conductive to electrophilic attack by the 2-hydroxyl group. In contrast, the 2-OH group of architecturally constrained nucleotides shows reduced nucleophilic reactivity. Sites of adduct formation are detected by RT-mediated primer extension which results in a pool of cDNA products whose lengths report the site of a modification. High-throughput SHAPE employing fluorescently labeled primers and automated capillary electrophoresis allows for resolution of 300-600 nt in a single Le Grice Lab capillary electrophoresis figure, 2 of 2electropherogram; it also provides the means to perform multiple experiments at once, i.e., several large RNAs can be mapped simultaneously using multiple primers and different experimental conditions. Subsequently, the resulting electropherogram, in which peak areas correspond with local flexibility at each individual nucleotide, is processed to yield a quantitative reactivity profile for the studied RNA. The obtained values are applied as pseudo-free-energy constraints, improving the accuracy of RNA secondary structure prediction algorithms. The expediency of this method and the availability of suitable data analysis tools make SHAPE acquiescent to a wide variety of problems, including analysis of viral genomes, intact messenger RNAs, and noncoding RNAs in distinct biological states.

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Last modified: 20 August 2013

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