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FdUMP[10] is a multimer of FdUMP, a suicide inhibitor of thymidylate synthase (TS), and was designed to bypass resistance to 5-fluorouracil (5FU). The aim of the study was to compare the effect of FdUMP[10] with 5FU and 5-fluoro-2-deoxyuridine (FUdR) in their efficacy to inhibit their target TS in resistant cells. Therefore cell lines FM3A/0, FM3A/TK- (deficient in thymidine kinase) and FM3A/TS- (deficient in thymidylate synthase) were used to determine TK dependency and specificity for TS inhibition. FdUMP[10] inhibited cell growth with greater potency than 5FU and FdUMP. Direct folate-based inhibitors Raltitrexed, GW1843U89 and Pemetrexed were also evaluated using these cell lines. In TK-deficient cells these folate-based inhibitors had greater potency than the fluoropyrimidines (FPs). Surprisingly, Pemetrexed even inhibited cell growth in TS-deficient cells. Incubation with nucleotidase and phosphatase inhibitors resulted in a reduction of cytotoxicity of FdUMP[10], indicating that the drug can be degraded outside the cells. In the TS in situ inhibition assay (TSIA) 24 h exposure of FM3A cells to 0.5 microM FdUMP and 0.05 microM FdUMP[10] decreased TSIA to 7 and 1% of control. Inhibition of nucleotidase and phosphatase activities reduced the effect of FdUMP[10], while the inhibitory effect was lower in cells lacking TK. FdUMP[10] can enter the cells intact, but also to some extent after dephosphorylation. In conclusion, FdUMP[10] can bypass resistance to FUdR by direct inhibition of TS.

It has been shown that DNA oligonucleotides composed, in part, of G repeat sequences can adopt G-quadruplex structures in the presence of specific metal ions. In this work, we use a combination of spectroscopic and calorimetric techniques to determine the spectral and thermodynamic characteristics of two DNA aptamers, d(G2T2G2TGTG2T2G2), G2, and d(G3T2G3TGTG3T2G3), G3; a sequence in the promoter region of the c-MYC oncogene, d(TG4AG3TG4AG3TG4A2G2), NHE-III; and the human telomere sequence d(AG3T2AG3T2AG3T2AG3), 22GG. The circular dichroism spectra of these oligonucleotides in the presence of K+ indicate that all form G-quadruplexes with G-quartets in an antiparallel arrangement (G2), in a parallel arrangement (NHE-III and 22GG), or in a mixed parallel and antiparallel G-quartet arrangement (G3). Melting profiles show transition temperatures, TM, above 45 degrees C that are independent of strand concentration, consistent with the formation of very stable intramolecular G-quadruplexes. We used differential scanning calorimetry to obtain complete thermodynamic profiles for the unfolding of each quadruplex. Subtracting the thermodynamic folding profiles of G2 from those of G3 yielded the following thermodynamic profile for the formation of a G-quartet stack: DeltaG degrees 20 = -2.2 kcal/mol, DeltaHcal = -14.6 kcal/mol, TDeltaScal = -12.4 kcal/mol, DeltanK+ = -0.3 mol of K+/mol, and DeltanW = 13 mol of H2O/mol. Furthermore, we used this profile to estimate the thermodynamic contributions of the loops and/or extra base sequences of each oligonucleotide in the G-quadruplex state. The average free energy contributions of the latter indicate that the incorporation of loops and base overhangs stabilizes quadruplex structures. This stabilization is enthalpy-driven and is due to base-stacking contributions.

FdUMP[10], a 10mer of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), the thymidylate synthase inhibitory metabolite of 5-fluorouracil (FU), is most closely correlated with the DNA topoisomerase I (Top1) inhibitor camptothecin in the National Cancer Institute COMPARE analysis, but not with FU. FdUMP[10] exhibits more potent antiproliferative activity than FdUMP or 5-fluoro-2'-deoxyuridine (FdU) and is markedly more active than FU. Camptothecin-resistant P388/CPT45 cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand breaks and cellular Top1-DNA complexes. Such complexes are also observed in response to FdUMP, FdU, raltitrexed, and FU. The FdUMP[10]-induced Top1-DNA complexes are not inhibited by the caspase inhibitor z-VAD-fmk and form independently of apoptotic DNA fragmentation, indicating that they do not correspond to apoptotic Top1-DNA complexes. In biochemical assay, Top1 is directly trapped at uracil and FdU misincorporation sites. We propose that FdUMP[10] damages DNA by trapping Top1 at uracil and FdU misincorporation sites resulting from thymidylate synthase inhibition and thymine depletion.

Hydrogen exchange rates of the imino protons of the thrombin-binding 15 mer DNA aptamer d(G(1)G(2)T(3)T(4)G(5)G(6)T(7)G(8)T(9)G(10)G(11)T(12)T(13)G(14)G(15)) in the presence of Sr(2+) were measured. In the temperature range 15-35 degrees C, the exchange rates of the eight iminos in the quadruplex core were not uniform, with the G(2), G(11) and G(15) iminos exchanging faster, the G(1), G(5), G(10) and G(14) iminos exchanging slower, and the G(6) imino exchanging at a medium rate. In the quadruplex G(1), G(5), G(10) and G(14) adopted syn glycosidic conformation, while G(2), G(6), G(11) and G(15) adopted anti-conformation. It was found that the four slowly exchanging iminos, which were all the syn-iminos, happened to be located in the TT loops that were not easy to open to the solvent. The anti-iminos exchanged faster, but the G(6) imino exchanged slower than other anti-iminos, because its hydrogen bond with the G(10)O6 was stabilized by the TGT loop. The fact that the G(6) imino exchanged at a faster rate than those syn-iminos in the TT loops suggested that the TGT loop was less stable than the TT loops. Unfolding mechanism for the quadruplex was thus proposed: The quadruplex first uncoupled the three base pairs: G(1)-G(15), G(2)-G(14) and G(5)-G(11), which were not protected by any loops. Then it opened the TGT loop. Finally, it opened the TT loops and the sequence became an unstructured random coil that exchanged with the quadruplex conformation. The conformational exchange between the quadruplex and random coil had been detected.

Thymidylate synthase (TS) is a well-validated target for cancer chemotherapy. TS was established as the principal target of the widely used anticancer drug 5-fluorouracil (5FU). The 5FU metabolite FdUMP forms a covalent complex with TS that is stabilized by 5-formyl tetrahydrofolate (leucovorin; LV). Numerous chemical strategies have been employed to develop novel TS inhibitors that are superior to 5FU/LV. 5FU is non-ideal as a TS-inhibitory drug because it is only inefficiently converted to FdUMP, while the remainder of the administered dose is converted to toxic metabolites. My laboratory has explored the utility of FdUMP[N] compounds (oligodeoxynucleotides comprised of FdUMP nucleotides) as FdUMP pro-drugs. FdUMP[N] compounds result in potent TS-inhibition, and display many advantages relative to 5FU/LV. A number of other chemical strategies have also been employed to develop pro-drugs, or metabolic precursors of FdUMP, and several of these strategies will be reviewed. In addition to chemical strategies to develop FdUMP pro-drugs, a number of chemical strategies have been devised to develop molecules that resemble the reduced folate co-factor required for TS catalysis. The synthesis of antifolates that have TS-inhibitory activity, such as Raltitrexed, has resulted in compounds that are effective and specific TS-inhibitors and, in some cases, have clinical potential. Chemical strategies that target TS mRNA for destruction are also being explored as potential chemotherapeutics. These diverse chemical approaches to control TS activity in tumor cells for the treatment of cancer will be reviewed.

The structure of thrombin-binding DNA aptamer complexed with a single Sr2+ ion (Sr2+:TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr2+:TBA complex is similar in topology, but distinct in structure, from that previously reported for the K+:TBA complex. The inter-tetrad distance of the Sr2+:TBA complex is 3.8 angstroms, or 0.7 angstroms larger than in the K+:TBA complex. This substantial difference can be attributed to a different binding site for Sr2+ in the Sr2+:TBA complex than for K+ in the K+:TBA complex. The Sr2+:TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr2+ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo., (Copyright 2004 Adenine Press)

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.

FdUMP[N] molecules and conjugates are much more effective at inhibiting the proliferation of human tumor cells than is the widely used anticancer drug 5-fluorouracil (5FU). We have evaluated the inhibition of thymidylate synthase (TS), the extent of DNA damage, cell cycle arrest, and the induction of apoptosis by FdUMP[10] and 5FU in the human colorectal cancer cell line HT29. The magnitude and duration of TS inhibition following exposure of HT29 cells to FdUMP[10] at 1 x 10(-8) M was greater than that which occurred following exposure of these cells to 5FU at 1 x 10(-6) M. FdUMP[10] exposure also resulted in much more extensive DNA damage to HT29 cells than occurred following exposure to 100-fold higher concentrations of 5FU. Although exposure of HT29 cells to both drugs resulted in S-phase arrest, more complete accumulation of cells in S-phase was achieved following FdUMP[10] exposure at much lower drug concentrations. FdUMP[10] was also much more effective at inducing apoptosis in HT29 cells than was 5FU. The results are consistent with FdUMP[10] being much more efficient that 5FU at inducing DNA damage that results in apoptotic cell death in colon cancer cells.

Nucleoside analogs such as 1-beta-D-arabinofuranosyl cytidine (AraC) and 2',2'-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2'-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3' endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.

Antimetabolites are a class of effective anticancer drugs that structurally resemble naturally occurring biochemicals and interfere in essential biochemical processes. In this review, the recent literature describing investigations of the structural and thermodynamic basis for the anticancer activity of three antipyrimidines [1-beta-D-arabinofuranosyl cytidine (AraC). 2',2'-difluoro deoxycytidine (dFdC), and 5-fluoro-2'-deoxyuridine (FdUrd)] is summarized. Our laboratory, and others, have shown that misincorporation of any of these three antipyrimidines into DNA perturbs the structure and decreases the stability of duplex DNA. These data are useful for rationalizing the effects of antipyrimidine misincorporation on the activities of proteins required for DNA replication and repair such as DNA topoisomerase 1 and DNA polymerases. The studies completed to date and summarized in this review demonstrate the utility of investigations into the structure-function relationships between antipyrimidine-substituted DNA complexed with DNA-modifying proteins for the purpose of understanding the basis for effective antipyrimidine cancer chemotherapy and the future design of novel anticancer drugs., (Copyright 2002 Wiley Periodicals, Inc. Biopolymers (Nucleic Acid Sci) 65: 180-189, 2002)

The NMR structure of the 3' stem-loop (3'SL) from human U4 snRNA was determined to gain insight into the structural basis for conservation of this stem-loop sequence from vertebrates. 3'SL sequences from human, rat, mouse and chicken U4 snRNA each consist of a 7 bp stem capped by a UACG tetraloop. No high resolution structure has previously been reported for a UACG tetraloop. The UACG tetraloop portion of the 3'SL was especially well defined by the NMR data, with a total of 92 NOE-derived restraints (about 15 per residue), including 48 inter-residue restraints (about 8 per residue) for the tetraloop and closing C-G base pair. Distance restraints were derived from NOESY spectra using MARDIGRAS with random error analysis. Refinement of the 20mer RNA hairpin structure was carried out using the programs DYANA and miniCarlo. In the UACG tetraloop, U and G formed a base pair stabilized by two hydrogen bonds, one between the 2'-hydroxyl proton of U and carbonyl oxygen of G, another between the imino proton of G and carbonyl oxygen O2 of U. In addition, the amino group of C formed a hydrogen bond with the phosphate oxygen of A. G adopted a syn orientation about the glycosidic bond, while the sugar puckers of A and C were either C2'-endo or flexible. The conformation of the UACG tetraloop was, overall, similar to that previously reported for UUCG tetraloops, another member of the UNCG class of tetraloops. The presence of an A, rather than a U, at the variable position, however, presents a distinct surface for interaction of the 3'SL tetraloop with either RNA or protein residues that may stabilize interactions important for active spliceosome formation. Such tertiary interactions may explain the conservation of the UACG tetraloop motif in 3'SL sequences from U4 snRNA in vertebrates.

Purpose: Gemcitabine-containing regimens are among standard therapies for the treatment of advanced non-small cell lung,pancreatic, or bladder cancers. Gemcitabine is a nucleoside analogue and its cytotoxicity is correlated with incorporation into genomic DNA and concomitant inhibition of DNA synthesis. However, it is still unclear by which mechanism(s) gemcitabine incorporation leads to cell death., Experimental Design: We used purified oligodeoxynucleotides to study the effects of gemcitabine incorporation on topoisomerase I (top1) activity and tested the role of top1 poisoning in gemcitabine-induced cytotoxicity in cancer cells., Results: We found that top1-mediated DNA cleavage was enhanced when gemcitabine was incorporated immediately 3' from a top1 cleavage site on the nonscissile strand. This position-specific enhancement was attributable to an increased DNA cleavage by top1 and was likely to have resulted from a combination of gemcitabine-induced conformational and electrostatic effects. Gemcitabine also enhanced camptothecin-induced cleavage complexes. We also detected top1 cleavage complexes in human leukemia CEM cells treated with gemcitabine and a 5-fold resistance of P388/CPT45 top1-deficient cells to gemcitabine, indicating that poisoning of top1 can contribute to the antitumor activity of gemcitabine., Conclusions: The present results extend our recent finding that incorporation of 1-beta-D-arabinofuranosylcytosine into DNA can induce top1 cleavage complexes [P. Pourquier et al. Proc. Natl. Acad. Sci. USA, 97: 1885-1890, 2000]. The enhancement of camptothecin-induced top1 cleavage complexes may, at least in part, contribute to the synergistic or additive effects of gemcitabine in combination with topotecan and irinotecan in human breast or lung cancer cells.

Thymidylate synthase (TS) is the molecular target of fluoropyrimidine (FP) chemotherapy, and novel anticancer drugs effective against TS-overexpressing tumors are required to treat patients with FP-refractory solid tumors. We have evaluated the inhibition of cell proliferation in vitro and antitumor activity in vivo of FdUMP[10], an oligodeoxynucleotide 10mer in which 5-fluorouracil (5-FU) is the only nucleobase. FdUMP[10] is a pro-drug of FdUMP, the TS inhibitory metabolite of FPs. FdUMP[10] was 338-fold more potent than 5-FU at inhibiting cell proliferation in the NCI 60 cell line screen. The antitumor activity of FdUMP[10] was compared to 5-FU using H-T29 xenografts in female CD-1 athymic (nu+/nu+) mice. Treatment with FdUMP[10] as a single agent (40 mg/kg/daily x 5, i.v.) delayed tumor growth and resulted in a smaller mean tumor size (T/C value = 51%, p<0.001 compared with the control group). Treatment with 5-FU (25 mg/kg/daily x 5, i.p.) had similar results as single agent FdUMP[10] (T/C value = 65%, p=0.238 compared with the FdUMP[10] treated group. Simultaneous treatment of tumor-bearing mice with both drugs (FdUMP[10] plus 5-FU) further delayed tumor growth (T/C value = 36%; p=0.003 relative to 5-FU). The results from the combined treatment group were not, however, statistically significant relative to the group receiving single agent FdUMP[10] treatment (p=0.059). Histological examination revealed systemic damage was limited to the colonic epithelium in all treatment groups and was least extensive with single agent FdUMP[10] compared to the other treatment groups. The data support the concept that FdUMP[10] is a useful prototype of a novel type of FP that is likely to be more efficacious than FPs in clinical use.

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.

Abstract A symposium co-organized by Professor William H. Gmeiner of Wake Forest University School of Medicine and Professor Moses Lee of Furman University was held March 30 and 31, 2001 on the campus of Wake Forest University School of Medicine. The Symposium was attended by many distinguished colleagues, friends and co-workers of Professor Lown who share his enthusiasm and passion for targeting DNA for treatment of human disease. The Symposium honored the formal "retirement" of Professor Lown who continues his active pursuit of scholarly activities and advancement of knowledge in a wide variety of intellectual interests.

Pre-mRNA splicing is one of the most complex and intricate processes in eukaryotic cell biology. Over the past decade, my laboratory has been interested in determining the structures of RNA components of the spliceosome, and in investigating how the structure, stability and dynamics of these RNA components are perturbed by nucleoside analog substitution. In particular, we have investigated the U4/U6 snRNA complex as a model system for understanding the biophysical basis for the RNA-mediated effects of the widely-used anticancer drug 5-fluorouracil ((5F)Ura). In this review, our studies that have provided novel information concerning the structure of U4 snRNA and its interactions with U6 snRNA and the Sm (or common) snRNA binding proteins are summarized. These studies have also quantified the structural and thermodynamic consequences of (5F)Ura in this model system. Our work to date provides the foundation on which future studies investigating the biophysical basis for spliceosomal assembly and for clarifying the mechanisms of anticancer drugs targeted at nucleic acid-mediated processes will be developed.

Gemcitabine (2'-deoxy-2',2'-difluorodeoxycytidine; dFdC) is a potent anticancer drug that exerts cytotoxic activity, in part, through incorporation of the nucleoside triphosphate dFdCTP into DNA and perturbations to DNA-mediated processes. The structure of a model Okazaki fragment containing a single dFdC substitution, [GEM], was determined using NMR spectroscopy and restrained molecular dynamics to understand structural distortions that may be induced in replicating DNA resulting from dFdC substitution. The electrostatic surface of [GEM] was also computed to determine how the geminal difluoro group of dFdC perturbs DNA electrostatics. The stability of [GEM] was investigated using temperature-dependent UV spectroscopy. dFdC adopted a C3'-endo conformation in [GEM] and decreased the melting temperature of the duplex by 4.3 degrees C. dFdC substitution did not decrease helical stacking among adjacent purines in the DNA duplex region. dFdC substitution substantially altered the electrostatic properties of the model Okazaki fragment, with increased electron density in the vicinity of the geminal difluoro group. The results are consistent with dFdC substitution altering the structural, electrostatic, and thermodynamic properties of DNA and interfering in DNA-mediated processes. Interference in DNA-mediated processes due to dFdC substitution likely contributes to the anticancer activity of dFdC.

The effects of native and non-native nucleotide substitution on the binding of Sm proteins to human U4 snRNA were investigated to determine if the Sm site was a likely target for the RNA-mediated effects of the anticancer drug 5-FU, and other nucleoside analogues. The Sm binding site of human U4 snRNA was prepared by in vitro transcription, and Sm protein binding was assessed using gel mobility shift assays. The U4:Sm RNA:protein complex was identified by immunoprecipitation with the Sm-specific Y12 antibody. The effects of 5-FU substitution were assessed by including FUTP in the in vitro transcription reactions. The effects of native nucleotide substitution were assessed by mutagenesis. Deletion mutants were used to assess the relative importance of the two stem-loops that flank the Sm binding site for protein binding. Point mutation (U-->G) to the 5'-Urd in the Sm site reduced Sm protein binding while similar point mutation to the 3'-Urd had a lesser effect. Mutation (U-->G) of all Urd in the Sm site completely inhibited Sm protein binding. The central stem-loop contributed significantly to Sm protein complex formation but the 3' stem-loop had little effect. Substitution of Urd by 5-fluorourdine (FUrd) did not inhibit Sm protein binding, but reduced the stability of the resulting complex. The results indicate that 5-FU, or other Uracil analogues, are unlikely to exert RNA-mediated effects through inhibition of Sm protein binding.

Current chemotherapy protocols that include fluoropyrimidines, such as 5-fluorouracil (5-FU), are limited by the development of chemoresistance during the course of treatment. Our laboratory has developed a novel class of fluoropyrimidines, FdUMP[N], that are oligodeoxynucleotides (ODNs) composed of some number, N, of 5-fluoro-2'-deoxyuridine-5'-O-monphosphate (FdUMP) nucleotides. Novel synthetic procedures are described that permit conjugation of folic acid to the 5'-OH of FdUMP[10] via a phosphodiester linkage using automated synthesis. The synthetic methods developed are generally applicable for ODN conjugation with folic acid. The folic acid conjugate FA-FdUMP[10] showed improved cytotoxicity toward human colorectal tumor cells (H630), and 5-FU-resistant colorectal tumor cells (H630-10). Enhanced cytotoxicity was observed for FA-FdUMP[10] relative to nonconjugated FdUMP[10] for cells grown under folate-restricted conditions, consistent with cellular uptake being, in part, receptor-mediated. Folate receptor alpha (FRalpha) mRNA was shown by RT-PCR to be overexpressed 26.3-fold in 5-FU-resistant H630-10 cells relative to H630 cells. Thus, FA-FdUMP[N] may prove useful for the treatment of 5-FU-resistant malignancies.

A 2200-ps molecular dynamics (MD) simulation of the U2 snRNA hairpin IV/U2B" complex was performed in aqueous solution using the particle mesh Ewald method to consider long-range electrostatic interactions. To investigate the interaction and recognition process between the RNA and protein, the free energy contributions resulting from individual amino acids of the protein component of the RNA/protein complex were calculated using the recently developed glycine-scanning method. The results revealed that the loop region of the U2 snRNA hairpin IV interacted mainly with three regions of the U2B" protein: 1) beta 1-helix A, 2) beta 2-beta 3, and 3) beta 4-helix C. U2 snRNA hairpin IV bound U2B" in a similar orientation as that previously described for U1 snRNA with the U1A' protein; however, the details of the interaction differed in several aspects. In particular, beta 1-helix A and beta 4-helix C in U2B" were not observed to interact with RNA in the U1A' protein complex. Most of the polar and charged residues in the interacting regions had larger mutant free energies than the nonpolar residues, indicating that electrostatic interactions were important for stabilizing the RNA/protein complex. The interaction was further stabilized by a network of hydrogen bonds and salt bridges formed between RNA and protein that was maintained throughout the MD trajectory. In addition to the direct interactions between RNA and the protein, solvent-mediated interactions also contributed significantly to complex stability. A detailed analysis of the ordered water molecules in the hydration of the RNA/protein complex revealed that bridged water molecules reside at the interface of RNA and protein as long as 2100 ps in the 2200-ps trajectory. At least 20 bridged water molecules, on average, contributed to the instantaneous stability of the RNA/protein complex. The stabilizing interaction energy due to bridging water molecules was obtained from ab initio Hartree-Fock and density functional theory calculations.

The design and synthesis of the lipophilic (9) and fluorescent (10) conjugates of a structural analogue of distamycin and their in vitro cellular localization studies are reported. Confocal laser scanning microscopy (CLSM) indicates that 10 rapidly enters human ovarian adenocarcinoma (SKOV-3) cells with principal uptake in mitochondria and uniform cytoplasmic distribution.

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PPII helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PPII helices led us to investigate whether proximal placement of a PPII helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PPII helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Escherichia coli. The fusion protein, SH3Hck : PPIIhGAP, folded spontaneously into a structure in which the PPII helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3Hck : PPIIhGAP fusion protein is useful for investigating SH3: PPII helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.

SH3 Domains provide interesting targets for investigations of protein structure and dynamics because of their compact size and importance for signal transduction. The present review summarizes recent research investigating SH3 domain structure and dynamics, the discovery of novel SH3 domains, the role of SH3 domains in disease, and progress in targeting SH3 domains for the development of novel therapeutics. Particular emphasis is placed on the unfolding/refolding characteristics of SH3 domains and the potential importance of these processes for regulation of signal transduction.

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3' stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3' stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U114, and the mismatched Ag91-G110 base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D1/D2 and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D3/B Sm protein sub-complex.

Gemcitabine (Gem) is a deoxycytidine analog that is effective against pancreatic cancer and other malignancies following conversion to the 5'-O-mono-, di- and tri-phosphate forms. We evaluated the cytotoxicity of GemMP[10], a novel multimeric form of 2'-deoxy-2',2"-difluorocytidine-5'-O-monophosphate (gemcitabine monophosphate) against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly-differentiated papillary (BHT-101) cancer. GemMP[10] decreased tumor cell growth at concentrations ranging from 1 to 50 nM. These concentrations were 5- to 10-fold lower than those required for inhibition of tumor cell growth by monomeric Gem. GemMP[10] cytotoxicity occurred via induction of apoptosis. Flow cytometric analysis of GemMP[10] treated cells revealed growth arrest in S-phase. Fas-antigen expression was increased in thyroid cancer cells treated with GemMP[10], whereas Fas-L and Bcl-2 expression were not significantly affected. These results demonstrated that GemMP[10] is a potent cytotoxic agent that serves to induce apoptosis in association with increased Fas expression in cultured thyroid cancer cell lines.

The binding of ligands to nucleic acids is of great interest for the control of gene expression and other nucleic acid mediated processes. We have evaluated the binding of several geometrically-constrained bis-distamycins to a model Okazaki fragment [OKA], or a DNA duplex having identical base sequence [DD], using gel-shift assays, optical spectroscopy and differential scanning calorimetry. In the case of covalent attachment of two distamycins to a central benzene ring, a similar binding profile was observed for [DD] as was observed for [OKA] (para binds [K(app) > 10(6) M(-1)], meta binds only weakly). For a central pyridyl ring, however, clear distinction between the binding to [DD] and binding to [OKA] was observed. While none of the three meta isomers having a central pyridyl ring bound [OKA], two of them (MT-17 and MT-12) bound [DD] [K(app) > 10(6) M(-1)]. These results demonstrate subtle differences in lexitropsin shape and placement of electronegative atoms may result in selective binding to a nucleic acid duplex based both on base sequence and chemical composition. Selective binding to DNA duplexes may be useful for designing ligands that regulate transcription, but do not interfere in other nucleic acid mediated processes.

The binding of ethidium bromide to a DNA hairpin (dU(5)-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1. 60x10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin, consistent with complex formation (1.22x10(-6) cm(2)/s at 26 degrees C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU(5)-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution.

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.

Okazaki fragments represent interesting targets for the design of anticancer drugs because of their selective occurrence during DNA replication, a process often elevated in aggressive malignancies. Structural studies have indicated a bend occurs in the helical axis at the junction region (JR) that joins the DNA duplex region (DDR) and the RNA-DNA hybrid duplex region (HDR) of model Okazaki fragments. To identify a structural motif that provides a shape complementary to the Okazaki fragment minor groove, we have investigated the binding of geometrically-constrained bis-distamycins to a model Okazaki fragment, [OKA], with a sequence derived from the genome of simian virus 40 (SV40). Both the JR and the DDR of [OKA] contain consecutive A/T base pairs that could accommodate distamycin binding. Of the six bis-distamycins selected for analysis, the two with a para configuration of the distamycins on the benzene or pyridine scaffold bound [OKA] tightly (Kd approximately 10(-6) M from gel-shift assays; Kd approximately 10(-8) M from deltaT(M)) while the four with a meta orientation did not bind. The two mono-distamycins studied also did not bind [OKA]. Molecular modeling of the complex between the para bis-distamycin MT-9 and [OKA] revealed MT-9 adopted an S- shape complementary to the minor groove of the model Okazaki fragment.

Interaction between 5-fluorouracil (5-FU) and FdUMP[10], a novel pro-drug formulation of the thymidylate synthase (TS) inhibitory nucleotide 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP), was investigated to evaluate the feasibility of using these two forms of fluorinated pyrimidine in combination chemotherapy regimens. 5-FU and FdUMP[10] are expected to differ in their relative intracellular distribution of active metabolites, and their combined administration may result in either a positive or a negative interactive effect. The dose-response behaviors of 5-FU and FdUMP[10] toward H630 and H630-10 (human colorectal tumor) cells were first investigated separately. Effects on cell viability were measured using an assay for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), while cytotoxicity and apoptosis were investigated using clonogenic and TUNEL assays, respectively. Exposure of H630 cells to concentrations of FdUMP[10] insufficient to inhibit cell proliferation as a single agent markedly increased the cytotoxicity of 5-FU. The results indicate that 5-FU and FdUMP[10] interact in a positive manner, and that combining these two forms of fluorinated pyrimidine may be clinically beneficial.

The efficacy of treatment with 5-Fluorouracil (5-FU) is limited, in part, by its inefficient conversion to 5-Fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP). We present data indicating that FdUMP[10], designed as a pro-drug for intracellular release of FdUMP, is cytotoxic as a consequence of uptake of the multimeric form. FdUMP[10] is stable in cell culture medium, with more than one-half of the material persisting as multimers of at least six nucleotides after a 48 h incubation at 37 degrees C. FdUMP[10] is more than 400 times more cytotoxic than 5-FU towards human colorectal tumor cells (H630). FdUMP[10] also has decreased toxicity in vivo, with doses as high as 200 mg/kg/day (qdx3) administered to Balb/c mice without morbidity, compared to a maximum tolerated dose of 45 mg/kg/day for 5-FU using the same protocol. FdUMP[10] shows reduced sensitivity to OPRTase- and TK-mediated drug resistance, relative to 5-FU and FdU, respectively, and is much more cytotoxic than 5-FU towards cells that overexpress thymidylate synthase. Thus, FdUMP[10] is less susceptible to resistance mechanisms that limit the clinical utility of 5-FU. The increased cytotoxicity, decreased toxicity in vivo, and reduced sensitivity to drug resistance of FdUMP[10], relative to 5-FU, indicates multimeric FdUMP is potentially valuable as an anti-neoplastic agent, either as a single agent, or in combination with 5-FU.

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.

The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.

The cytotoxicity of the 10mer ODN FdUMP[10] towards human colorectal tumor cells was evaluated using a clonogenic assay. FdUMP[10] was more than 100-fold more active than 5-FU at inhibiting colony formation of H630 cells. FdUMP[10] was also evaluated for cytotoxicity in the NCI 60 cell line screen, and showed markedly improved activity relative to 5-FU against numerous tumor cell lines. The in-vivo tolerance of FdUMP[10] is more than three-fold greater per mole fluorinated pyrimidine, than 5-FU.

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains., (Copyright 1999 Academic Press.)

Okazaki fragments occur as intermediates during lagging strand DNA replication. Alterations in Okazaki fragment structure may contribute to the anticancer activities of nucleoside analogues such as cytarabine, a potent anti-leukemic agent that inhibits lagging strand replication. We have determined the solution structures for two model Okazaki fragments, [OKA] and [ARAC]. These sequences are derived from a frequent initiation site for primase during replication of the SV-40 viral genome. The sequence of [ARAC] differs from [OKA] only by substitution of cytarabine for one deoxycytidine. The structure of each model Okazaki fragment was elucidated using NMR spectroscopy and restrained molecular dynamics simulations. The solution structures of [OKA] and [ARAC] each consist of two distinct domains: a DNA duplex region (DDR) and an RNA-DNA hybrid duplex region (HDR). The DDR of [OKA] adopts geometry similar to B-form except for variations in helical parameters, especially twist and roll, which occur in the purine tract, increasing base overlap among the five consecutive purines. The helical axes for the DDR and HDR of [OKA] are bent 22 degrees relative to one another. Although the local structures for the DDR and HDR of [ARAC] are similar to those in [OKA] (root-mean-square deviation (rmsd) approximately 0.8, 1.7 A), the bending at the junction is different (41 degrees for [ARAC] vs 22 degrees for [OKA]). Increased helical bending of cytarabine-substituted Okazaki fragments may contribute to the propensity of cytarabine to inhibit elongation of the lagging strand during DNA replication, and in effecting anticancer activity.

Decreased dynamic motion in the peptide backbone of proteins may accompany ligand binding and influence the thermodynamic and kinetic stability of the resulting complexes. We have investigated the diffusional behavior and backbone dynamics of the free and phosphopeptide (EPQpYEEIPIYL) complexed Hck SH2 domain using NMR spectroscopy. Both the free domain and its phosphopeptide complex self-associate at higher protein concentrations. Diffusional measurements and surface analysis indicate that charged side-chain groups are probably responsible for self-association. Higher order aggregation, such as trimer and tetramer, also occurs at elevated protein concentrations. Dynamic motion in the peptide backbone of Hck SH2 was determined from 15N relaxation data fit using extended model-free parameters. The rotational correlation time (taum) for uncomplexed Hck SH2 was 6.8 ns while taum for peptide-bound Hck SH2 was 7.6 ns. Generalized order parameters (S2) increased for most residues upon binding of the phosphopeptide, consistent with peptide binding restricting motion of the NH bond vectors on the picosecond time scale. These studies suggest that complexation increases internal order in Hck SH2 and that internal dynamic motions contribute to the activation of Src-family kinases in vivo.

Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. The effects of cytarabine substitution on the structural and thermodynamic properties of a model Okazaki fragment were investigated using UV hyperchromicity and 1H NMR spectroscopy to determine how cytarabine alters the physicochemical properties of Okazaki fragments that are intermediates during DNA replication. Two model Okazaki fragments were prepared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together with its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substitution for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition for each model Okazaki fragment were calculated from the concentration dependence of the T m at 260 nm. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(-5) M. The free energy for the duplex to single strand transition was 1.2 kcal/mol less favorable for the cytarabine-substituted Okazaki fragment relative to the control at 37 degrees C. Analysis of the temperature dependence of the imino1H resonances for the two duplexes demonstrated that cytarabine specifically destabilized the DNA:DNA duplex portion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okazaki fragments in vivo .

SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain., (Copyright 1998 Academic Press Limited.)

NMR spectroscopy has been shown to be useful in determining the structures of nucleic acid fragments in solution. Over the last several years NMR spectroscopy, in conjunction with restrained molecular dynamics, has been employed to understand the 3D structures of a number of anticancer drugs and to rationalize their DNA binding behavior. In this review we address the methodologies used most frequently to determine nucleic acid structures in solution. In subsequent sections, we examine how these methods have been applied to rationalize the activities of a number of anticancer agents that target duplex DNA such as cisplatin, bleomycin and calicheamicin. Non-duplex DNA and RNA also represent interesting nucleic acid targets for anticancer drug design and applications of solution NMR spectroscopy to understanding the structures of these types of molecules (e.g. Okazaki fragments, DNA tetraplexes) are also reviewed. In the final sections, advances in NMR methodologies (e.g. linear prediction, superconducting probes) that are likely to impact the research conducted in this area are reviewed. The success of NMR spectroscopy in understanding the structural basis for clinically useful anticancer drugs bodes well for future applications of this methodology not only in rationalization of existing biological activity, but in the design of novel agents that will be useful in treating neoplastic disease.

Protein unfolding on a fast time scale (milliseconds-minutes) has been widely reported, but slower unfolding events (10 min-hours) have received less attention. Amide hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the unfolding dynamics of the hematopoietic cell kinase (Hck) SH3 domain. Analysis of mass spectra after deuterium exchange into intact Hck SH3 indicates a cooperative unfolding event involving 24-61% of the domain and occurring with a half-life of approximately 20 min under physiological conditions. To identify the unfolding region, SH3 was incubated in D2O and proteolytically fragmented into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in several regions, including the C-terminal half of the RT-loop and a beta-sheet flanking the binding site. Binding of a prolyl-rich segment from the HIV Nef protein slows unfolding by a factor of 3. Further analysis yields a KD of 25 microM for the Nef peptide. These results demonstrate that an inherent flexibility in the SH3 domain may assist interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. The combination of hydrogen exchange and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.

The hematopoietic cellular kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed predominantly in granulocytes, monocytes and macrophages. Recent observations suggest that Hck may be activated in HIV-infected macrophages and in chronic myelogenous leukemia cells that express Bcr-Abl. In order to increase our understanding of the structural basis for regulation of Hck activity under normal and pathological conditions, we have solved the solution structure of the uncomplexed Hck SH2 domain using NMR spectroscopy. A novel procedure that uses intraresidue HN-H alpha distances as references for converting NOE intensities into distance restraints has been described. A total of 1757 significant experimental restraints were derived from NMR spectroscopic data including 238 medium-range and 487 long-range distance restraints and 177 torsion angle restraints. These restraints were used in a simulated annealing procedure to generate 20 structures with the program DYANA. Superimposition of residues 5-104 upon the mean coordinate set yielded an average atomic rmsd values of 0.42 +/- 0.08 A for the N,C alpha,C' atoms and 0.81 +/- 0.08 A for all heavy atoms. Rmsd values for those residues in the regions of ordered secondary structure were 0.27 +/- 0.04 A for the N,C alpha,C' atoms and 0.73 +/- 0.06 A for all heavy atoms.

The structures and stabilities of three RNA duplexes that differed only in the position of 5-fluorouridine (FUrd) substitution were elucidated using NMR spectroscopy and UV hyperchromicity studies to determine if FUrd substitution altered the structure or stability of RNA duplexes that contained G-U base pairs. The duplexes investigated corresponded to the region of the U4-U6 snRNA complex that contained the 5' terminus of U4 snRNA. The control duplex contained a G-U wobble base pair and also a G-A mismatched base pair. FUrd was substituted in one duplex at the G-U wobble base pair and in the second duplex at an A-U base pair adjacent to the wobble base pair. FUrd substitution slightly destabilized the duplex that contained a G-FU base pair but stabilized the duplex that contained an A-FU base pair. NOESY spectra were used to determine interproton distances, and these distance constraints were used in a restrained molecular dynamics protocol to determine the three-dimensional structures of these RNA duplexes. Analyses of helical parameters, backbone torsion angles, and rms deviations between the final structures revealed no systematic differences due to FUrd substitution in RNA duplexes that contained G-U base pairs. The G-FU base pair adopted wobble geometry, while the G-A mismatch formed a sheared base pair. NOESY spectra in H2O solution revealed the imino 1H from FUrd exchanged more rapidly with solvent than did the Urd imino 1H but did not show the G-FU base pair adopted an ionized structure. Reduced stacking occurred for the G-FU base pair relative to the G-U base pair in the time-averaged structure, and this, rather than ionization of the base pair, was responsible for the slight destabilization of the duplex that contained the G-FU base pair.

The hematopoietic cellular kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases and participates in signal transduction events regulating the growth, differentiation and function of phagocytes. The secondary structure of the SH2 domain for Hck was determined for a 13C/15N-enriched sample using multi-dimensional NMR spectroscopy. The secondary structure for the domain was determined from chemical shift indices [1H alpha, 13C alpha and 13C'], sequential NOEs [d(alphaN)(i, i+1) and d(NN)(i, i+1)], and 3J(alphaN) scalar coupling constants. The Hck SH2 domain consists of two alpha-helices and seven beta-strands. Complementary strands of beta-sheets were identified from long-range NOEs using a novel 3D, 13C/15N-edited HMQC-NOESY-(HCACO)NH experiment that correlated 1H alpha resonances between beta-strands. The secondary structure for Hck SH2 is similar to that predicted from the sequence alignment of the Src-family protein tyrosine kinases.

1H NMR and molecular modeling studies of the 5' stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5' side and a single bulged A on the 3' side. Both stems also had bulged A residues. 1H NMR experiments performed on solutions of the 35 mer stem-loop, the stem region, and the loop region confirmed the 35 mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.

The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution: (5' dGCGAAUUCGC)2, (5' dGCGAAUFCGC)2, (5' dGCGAAFUCGC)2, and (5' dGCGAAFFCGC)2. The corresponding oligoribonucleotide sequences (5' rGCGAAUUCGC)2, (5' rGCGAAUFCGC)2, (5' rGCGAAFUCGC)2, and (5' rGCGAAFFCGC)2 were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA-RNA and RNA-protein recognition.

A 3D NOESY-(HCACO)NH experiment is described that transfers NOEs from 1H alpha to the backbone 1Hn in the succeeding residue for detection. Using this strategy, NOEs involving 1H alpha protons that resonate exactly at the water frequency can be detected. NOEs from an overlapping 1H alpha proton that is attached to degenerate 13C alpha can also be resolved. The performance of this approach is demonstrated for the 13C-/15N-labeled Hck/SH2 dissolved in H2O.

Pulsed field gradients were incorporated into the HCACO experiment for acquiring spectra on isotopically enriched protein samples dissolved in H2O. Excellent water suppression and spectral quality were achieved using the modified pulse sequence (gd-HCACO), as demonstrated for a 13C-/15N-labeled sample of the SH2 domain from the hematopoietic cellular kinase dissolved in 90% H2O/10% D2O. Strong correlations for all residues were observed in the gd-HCACO spectrum, even for residues having alpha-protons resonating exactly at the H2O frequency. The HCACO-TOCSY experiment was modified to correlate intraresidue 13C alpha (rather than 1H alpha), carbonyl (13C'), and aliphatic side-chain protons [(H)CACO-TOCSY]. Pulsed field gradients were also incorporated into the (H)CACO-TOCSY experiment for water suppression.

Nuclear magnetic resonance spectroscopists are increasingly utilizing chemical shifts to characterize the secondary structure of proteins. The present study addresses the effects that the positively charged amino group at the N-terminus of a peptide has on 1HN and 1HC alpha chemical shifts along the chain. This information is necessary for interpreting chemical shift data for proteins and/or for peptides that are used as models for protein structure. The chemical shifts for the 1H resonances of four peptides that differ only in the location of their N-terminii are assigned using two-dimensional nmr spectroscopy. The peptides have sequences derived from the beta subunit of the glycoprotein hormone human chorionic gonadotropin (hCG-beta). Comparison of the 1HN and the 1HC alpha chemical shifts for residues common to all four peptides reveals downfield shifts for 1HN and the 1HC alpha resonances within three residues of the N-terminus compared with chemical shifts in the interior of the peptide. The magnitude of the downfield shift is larger for resonances nearer the N-terminus. Quantum mechanical calculations of the 1HN and 1HC alpha chemical shifts in peptides constructed with six alanine units also predict a significant terminus effect. The calculations agree both qualitatively and quantitatively with the experimental data. The inductive nature of the end effect is confirmed in the calculations by Mulliken population analysis. End effects should be taken into account in determining protein secondary structures from chemical shifts.