Barbara L. Gaffney

Structural basis of HIV-1 resistance to AZT by excision.

Human immunodeficiency virus (HIV-1) develops resistance to 3′-azido-2′,3′-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3′ end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase–double-stranded DNA (RT–dsDNA)–AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT–dsDNA–AZTppppA; AZTr RT–dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg72 were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg72 were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.

A new strategy for the protection of deoxyguanosine during oligonucleotide synthesis

The protection of the 6-oxo group of deoxyguanosine with the 2-trimethylsilylethyl (2), phenylthioethyl (3), 4-nitrophenylthioethyl (4), 4-nintrophenethyl (5), and cyanoethyl (6) groups is described. Each protecting group is introduced in good yield and is cleaved under mild conditions. Compatibility with the various approaches to oligonucleotide synthesis is discussed.

Differential analogue binding by two classes of c-di-GMP riboswitches.

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism bacteria have evolved to sense environmental cues and translate these signals into phenotypic changes uses the second messenger signaling molecule, cyclic diguanosine monophosphate (c-di-GMP). In addition to several classes of protein receptors, two classes of c-di-GMP-binding riboswitches (class I and class II) have been identified as downstream targets of the second messenger in this signaling pathway. The crystal structures of both riboswitch classes bound to c-di-GMP were previously reported. Here, we further investigate the mechanisms that RNA has evolved for recognition and binding of this second messenger. Using a series of c-di-GMP analogues, we probed the interactions made in the RNA-ligand complex for both classes of riboswitches to identify the most critical elements of c-di-GMP for binding. We found that the structural features of c-di-GMP required for binding differ between these two effectors and that the class II riboswitch is much less discriminatory in ligand binding than the class I riboswitch. These data suggest an explanation for the predicted preferential use of the class I motif over the class II motif in the c-di-GMP signaling pathway.

One-Flask Syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] Thiophosphate Analogues

An integrated set of reactions and conditions that allow an eight-step one-flask synthesis of the protected derivatives of c-di-GMP and the [R(p),R(p)] and [R(p),S(p)] thiophosphate analogues is reported. Deprotection is also carried out as a one-flask procedure, with the final products isolated by crystallization from the reaction mixture. Chromatography is only used for separation of the thiophosphate diastereomers.

Synthesis of O-6-alkylated deoxyguanosine nucleosides

Abstract A general route for synthesis of 6- O -alkyl-2∝deoxyguanosine nucleosides is described. The key step is conversion of the 6- O -TPS derivative 2 to the 6-trimethylamino compound 3 . The trimethylamino group is readily displaced by alcohols in the presence of DBU. Using this route the 6- O -methyl, ethyl and n-butyl 2∝-deoxyguanosine derivatives 5a-c have been prepared in excellent overall yields.

Synthesis of 6-substituted 2′-deoxyguanosine derivatives using trifluoroacetic anhydride in pyridine

Abstract Trifluoroacetic anhydride at 0° C reacts with a pyridine suspension of deoxyguanosine to generate a polar intermediate, presumably the corresponding 6-pyridyl derivative. The reaction is complete in less than 15 minutes, and is not accompanied by degradation. From this intermediate a variety of 6-substituted deoxyguanosine derivatives can be obtained, some in excellent yields.

Large-scale oligonucleotide synthesis by the H-phosphonate method

Abstract The H-phosphonate method is capable of giving good yields, on scales in the area of 14 μmole, with as little as 2eq of monomer. Under these conditions, however, capping was found to be necessary. Cyanoethyl H-phosphonate has been introduced for this purpose and shown to be effective.

H-phosphonate oligonucleotide synthesis on a polyethylen glycol/polystyrene copolymer

Abstract The scale of oligonucleotide synthesis can be increased by a factor of 2–3 by using a polyethylene glycol/polystyrene copolymer support in place of controlled-pore glass. This support allows synthesis on a scale of 30 μmole in a standard 10–15 μmole cartridge. In addition, the recovery and reuse of H-phosphonate monomers are reported and a system for automation of the trityl assay is described.

Covalent carcinogenic O6-methylguanosine lesions in DNA structural studies of the O6meG·A and O6meG·G interactions in dodecanucleotide duplexes

High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes.