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 dNTPand 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.The development of effective anti-HIV drugs and their use in combination therapies has been estimated to have saved more than 3 million years of life 1 . However, drug therapies do not cure HIV infections, and drug therapy is life-long. Prolonged therapies lead to problems with drug toxicity and drug resistance. Understanding the molecular mechanisms of resistance is important for developing more effective drugs and treatment strategies. Many anti-AIDS drugs © 2010 Nature America, Inc. All rights reserved.Correspondence should be addressed to E.A. (arnold@cabm.rutgers.edu).. 4 Present address: Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, Missouri, USA 5 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS X.T. carried out the research, did analysis and helped with writing the manuscript. K.D. advised on crystallographic studies and did analysis, writing and editing of the manuscript. Q.H., J.D.B., X.H., B.L.G. and R.A.J. contributed special reagents. A.D.C. and Y.V.F. carried out parts of the research. P.L.B. contributed special reagents and manuscript editing. S.H.H. carried out analysis and manuscript writing and editing. S.G.S. designed the research and advised on experiments and manuscript editing. E.A. designed the research, supervised the project and did analysis, writing and editing of the manuscript. METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/. Accession codes. Protein Data Bank: Coordinates and structure factors for AZTr RT-dsDNA-AZTppppA', wild-type RT-dsDNAAZTppppA', AZTr reverse transcriptase N-site and P-site complexes, and apo AZTr reverse transcriptase have been deposited with accession codes 3KLE, 3KLF, 3KLG, 3KLH and 3KLI, respectively. Note: Supplementary information is available on the Nature Structural Molecular Biology website. COMPETING FINANCIAL I...

[reaction: see text] We report a one-flask route for the synthesis of dinucleoside tetra- and pentaphosphates, in isolated yields of 50-85%. This route relies on a mixture of P(III) and P(V) chemistries, using phosphitylation of a protected nucleoside with 2-chloro-4H-l,3,2-benzo-dioxaphosphorin-4-one (salicylchlorophosphite), followed by sequential reaction with inorganic pyrophosphate and a nucleoside 5' mono- or diphosphate.

Abstract15 N NMR chemical shift changes in the presence of Mg(H 2 O) 6 2+ , Zn 2+ , Cd 2+ , and Co(NH 3 ) 6 3+ were used to probe the effect of flanking bases on metal binding sites in three different RNA motifs. We found that: for GC pairs, the presence of a flanking purine creates a site for the soft metals Zn 2+ and Cd 2+ only; a GG·UU motif selectively binds only Co(NH 3 ) 6 3+ , while a UG·GU motif binds none of these metals; a 3′ guanosine flanking the adenosine of a sheared GA·AG pair creates an unusually strong binding site that precludes binding to the cross-strand stacked guanosines within the tandem pair.

We report an efficient, one-flask route for synthesis of AZTpSpCX2ppSA and AZTpSpCX2ppSAZT, where X=H and X=F. This route makes use of the differential susceptibility to oxidation of H-phosphonate mono- and diesters, to allow a series of sequential reactions without requiring isolation of intermediates. These compounds are hydrolysis-resistant versions of the AZTppppA that results from excision of AZT by AZT-resistant HIV reverse transcriptase (RT). This family of compounds may therefore be useful in further study of the AZT excision reaction, as well as in drug design.