Biomedical subjects
Publications and source records attributed to J Feigon.
Solution structures of RNA aptamers for FMN, ATP, arginine, and citrulline reveal how oligonucleotides can fold to form selective binding pockets for biological cofactors and amino acids. These structures confirm old ideas and provide new insights about three-dimensional structures of nucleic acids and their possible role in chemical reactions.
BACKGROUND: The protegrins are a family of arginine- and cysteine-rich cationic peptides found in porcine leukocytes that exhibit a broad range of antimicrobial and antiviral activities. They are composed of 16-18 amino-acid residues including four cysteines, which form two disulfide linkages. To begin to understand the mechanism of action of these peptides, we set out to determine the structure of protegrin-1 (PG-1). RESULTS: We used two-dimensional homonuclear nuclear magnetic resonance spectroscopy to study the conformation of both natural and synthetic PG-1 under several conditions. A refined three-dimensional structure of synthetic PG-1 is presented. CONCLUSIONS: Both synthetic and natural protegrin-1 form a well-defined structure in solution composed primarily of a two-stranded antiparallel beta sheet, with strands connected by a beta turn. The structure of PG-1 suggests ways in which the peptide may interact with itself or other molecules to form the membrane pores and the large membrane-associated assemblages observed in protegrin-treated, gram-negative bacteria.
In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures.
BACKGROUND: Oligonucleotides containing the guanine-rich telomeric sequence of Oxytricha chromosomes (dT4G4) have previously been shown to form DNA quadruplexes comprising guanine quartets stabilized by cations. Two different structures have been reported for both d(G4T4G4) (Oxy1.5) and d(G4T4G4T4G4T4G4) (Oxy3.5). RESULTS: Here we present the solution structure of a uracil- and inosine-containing derivative of Oxy3.5, d(G4TUTUG4T4G4UUTTG3I) (Oxy3.5-U4128), determined using two-dimensional 1H and 31P NMR techniques. This oligonucleotide forms a unimolecular quadruplex that is very similar to the dimeric Oxy1.5 solution structure, in that it contains a loop spanning the diagonal of an end quartet. The groove widths, strand polarities, and positions of the syn bases along the G4 tracts and within the quartets are all as reported for Oxy1.5. The first and third pyrimidine tracts form parallel loops spanning a wide groove and a narrow groove respectively. CONCLUSIONS: Both Oxy3.5 and Oxy3.5-U(4)128 form unimolecular quadruplexes in solution with a diagonal central T4 loop. These results conflict with those reported for d(G4TUTUG4TTUUG4UUTTG4) in solution, in which the central loop spans a wide groove.
The reverse transcriptase (RT) of HIV-1 is a plausible target for therapeutic agents aimed at inhibiting propagation of the virus. We have used "irrational drug design", that is, combinatorial chemistry with oligonucleotide libraries, to identify high-affinity ligands aimed at HIV-1 RT. The methodology, termed SELEX (systematic evolution of ligands by exponential enrichment), was employed with a single-stranded DNA library. The selected ssDNA ligands bind HIV-1 RT with Kd values as low as 1 nM and inhibit the RNA-dependent DNA-polymerase activity of the enzyme with Ki values as low as 0.3 nM. We also demonstrate the high specificity of one ligand able to selectively discriminate between the reverse transcriptases of HIV-1, AMV, and MMLV. These ssDNA molecules may be useful as inhibitors or as models for the design of small molecule inhibitors of HIV-1 RT in vivo.
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Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.
The structure formed by the DNA oligonucleotide d(G3T4G3) has been studied by one- and two-dimensional 1H NMR spectroscopy. In NaCl solution, d(G3T4G3), like d(G4T4G4) (Oxy-1.5), forms a dimeric quadruplex with the thymines in loops across the diagonal of the end quartets. Unlike Oxy-1.5, the dimer is not symmetric, and both monomer strands are observed in NMR spectra. Three quartets are formed from the GGG tracts. Glycosidic conformations of the guanines are 5'-syn-syn-anti-(loop)-syn-anti-anti in one strand and 5'-syn-anti-anti-(loop)-syn-syn-anti in the other strand. Thus, the stacking of the quartets (tail-to-tail, head-to-tail) is unlike all previously described fold-back (tail-to-tail, head-to-head) and parallel-stranded (head-to-tail, head-to-tail) quadruplexes.
Triostin A, a naturally occurring quinoxaline antibiotic that contains N-methyl groups on the valine and cysteine residues, binds sequence specifically to DNA at NCGN sites. [N-MeCys3,N-MeCys7]-TANDEM (CysMeTANDEM), a synthetic quinoxaline antibiotic, differs in its chemical structure from triostin A only at the valine residues, which contain no N-methyl substituents. CysMeTANDEM has a sequence specificity different from triostin A, binding specifically to DNA at NTAN sites. To understand the factors that determine the sequence specificity of these quinoxaline antibiotics, the solution structure of a 1:1 complex of triostin A with the DNA hexamer [d(GACGTC)]2 has been determined using NMR-derived distance and dihedral angle restraints. The solution structure of the triostin A-[d(GACGTC)]2 complex is compared directly to the solution structure of a 1:1 complex of CysMeTANDEM with [d(GATATC)]2 and is also compared to the crystal structure of 2:1 complex of triostin A with [d(CGTACG)]2. Triostin A binds to [d(GACGTC)]2 as a bis-intercalator around the CpG step, and the peptide ring of the drug binds in the minor groove of the DNA. The central C.G base pairs of the complex are underwound with an average helical twist angle of approximately -9.0 degrees and buckle inward by about 25 degrees. There are intermolecular hydrogen bonds between each of the Ala NH and the GN3 protons of the CpG binding site. Similar structural features are observed in the solution structure of the CysMeTANDEM-[d(GATATC)]2 complex. However, in the structure of the triostin A-[d(GACGTC)]2 complex, two intermolecular hydrogen bonds between each of the Ala CO oxygens of the drug and the 2-amino protons of guanine are observed. These hydrogen bonds do not form in the CysMeTANDEM-DNA complex. Instead, CysMeTANDEM contains two intramolecular hydrogen bonds between the Ala CO atoms and the Val amide protons, making the Ala CO atoms unavailable to form two intermolecular hydrogen bonds. The role of these intermolecular hydrogen bonds in the CpG specificity of triostin A is discussed.
The binding of CysMeTANDEM and triostin A to DNA containing a CpI step has been studied by one- and two-dimensional 1H NMR spectroscopy. CysMeTANDEM binds sequence specifically to CpI steps as well as TpA steps as a bis-intercalator with the peptide backbone in the minor groove of the DNA. Only nonspecific, nonintercalative binding is observed between triostin A and DNA containing a CpI step. Comparison of the CysMeTANDEM-[d(GGACITCC)]2 complex to the CysMeTANDEM-[d(GGA-TATCC)]2 complex indicates that the structures of both complexes are very similar. However, CysMeTANDEM binds less tightly to [d(GGACITCC)]2 than to [d(GGATATCC)]2. The NMR evidence presented provides molecular insight into the role of stacking interactions and hydrogen bonding between the drug and the DNA in the sequence-specific binding of CysMeTANDEM to TpA sites and of triostin A to CpG sites.
1H NMR studies of a 30-nucleotide RNA oligonucleotide (RBE3), which contains a high-affinity binding site for Rev of the HIV-1 Rev responsive element (RRE), two derivatives of RBE3 (RBE3AA and RBE3-A), and the complex of RBE3 with peptides derived from the RNA binding domain of HIV-1 Rev, are presented. The high-affinity binding site of the RRE consists of an asymmetric internal loop and surrounding Watson-Crick base pairs. In the wild-type RRE, one of the stems is closed by a loop; this is replaced in REB3 by the stable UUCG tetraloop. NOE data suggest that the internal loop of the free RNA contains structural features that have been predicted on the basis of in vitro selection experiments [Bartel, D.P., et al. (1991) Cell 67, 529-536]. The structural features include a Gsyn.Ganti base pair, a Ganti.Aanti base pair, and a looped out U. When the Rev peptide is bound to the RNA, the base pairs in the internal loop appear to be stabilized, although the RNA chemical shifts indicate that the RNA conformation undergoes some changes when bound by Rev peptide.
BACKGROUND: Telomeres, the structures at the ends of linear eukaryotic chromosomes, are essential for chromosome replication and stability. The telomeres of the unicellular ciliate Oxytricha contain a 3' single strand overhang composed of two repeats of the telomere repeat sequence d(TTTTGGGG). It has been proposed that oligonucleotides containing this repeat can form DNA quadruplexes via hydrogen bonding of the guanines into quartets. Such structures may be relevant to the biological function of the telomere, and in G-rich sequences elsewhere in the genome. RESULTS: We have previously determined from solution NMR data that the Oxy-1.5 Oxytricha repeat oligonucleotide d(GGGGTTTTGGGG) dimerizes to form an intermolecular quadruplex composed of four guanine quartets and with the thymines in loops across the diagonal at opposite ends of the quadruplex. We report here the refined solution structure of Oxy-1.5. This structure is compared with the previously published crystal structure of the same oligonucleotide. CONCLUSIONS: Oxy-1.5 forms a well-defined, symmetrical structure with ordered thymine loops. Both the solution and crystal structures of Oxy-1.5 are quadruplexes with alternating syn and anti glycosyl conformation of guanines along each strand of the helix and have thymine loops at opposite ends. However, the topology of the two structures is fundamentally different, leading to significant structural differences. A topological pathway for the formation and interconversion of the two structures is proposed.
The DNA oligonucleotide d(GGTTGGTGTGGTTGG) (thrombin aptamer) binds to thrombin and inhibits its enzymatic activity in the chain of reactions that lead to blood clotting. Two-dimensional 1H NMR studies indicate that the oligonucleotide forms a folded structure in solution, composed of two guanine quartets connected by two T-T loops spanning the narrow grooves at one end and a T-G-T loop spanning a wide groove at the other end. We present the assignment strategy used, methods for the structure determination, and the refined three-dimensional structure of the thrombin aptamer. The initial structures were generated by metric matrix distance geometry using distance and dihedral bond angle constraints from NOE and coupling constants, respectively, and refined by restrained molecular dynamics and direct NOE refinement. Knowledge of the three-dimensional structure of this thrombin aptamer may be relevant for the design of improved thrombin-inhibiting anti-coagulants with similar structural motifs.
The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studied by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1' and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RN oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1' and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.
The structure formed from the DNA oligonucleotide d(G4T4G4) (Oxy-1.5), which contains the Oxytricha telomere repeat T4G4, has been investigated by two-dimensional 1H and 31P NMR spectroscopy. Sequence-specific assignments have been obtained for the 1H and 31P resonances, using a combination of methods including comparisons to the inosine- and uracil-containing derivatives d(G4T4G3I) and d(G4UT3G4). The oligonucleotide forms a symmetrical bimolecular G-quadruplex with four G-quartets and thymine loops at opposite ends of the G-quartets. Guanines are alternatively syn and anti along each "strand" and all of the thymines are anti. The thymines loop diagonally across the G-quartet, resulting in a structure in which adjacent strands are alternately parallel and antiparallel and the glycosidic torsion angles are syn-syn-anti-anti around each G-quartet. There are three different types of grooves, a wide, a narrow, and two medium grooves. A diagonally looped quadruplex is formed in the presence of both Na+ and K+ counterions. The model structure of Oxy-1.5 is compared to the recently published crystal structure of Oxy-1.5 (Kang et al., 1992), which contains many of the same features as those found in solution but differs in that the thymines loop across an edge of the G-quartet.
We have used two-dimensional 1H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355, 564-566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.
[N-MeCys3,N-MeCys7]TANDEM (CysMeTANDEM) is an octadepsipeptide quinoxaline antibiotic that binds specifically by bisintercalation to double-stranded DNA at NTAN sites [Addess, K. J., Gilbert, D. E., Olsen, R. K., & Feigon, J. (1992) Biochemistry 31, 339-350; Addess, K. J., Gilbert, D. E., & Feigon, J. (1992) in Structure and Function Volume 1: Nucleic Acids (Sarma, R. H., & Sarma, M. H., Eds.) pp 147-164, Adenine Press, Schenectady, NY]. We have determined the three-dimensional structure of a complex of CysMeTANDEM and the DNA hexamer [d(GATATC)]2 using two-dimensional 1H NMR derived NOE and dihedral bond angle constraints. This is the first structure of a TpA-specific quinoxaline antibiotic in complex with DNA. Initial structures of the complex were generated by metric matrix distance geometry followed by simulated annealing. Eight of these structures, refined by restrained molecular dynamics, energy minimization, and NOE-based relaxation matrix refinement, have an average pairwise RMSD of 1.11 A for all structures, calculated using all heavy atoms of the drug and the DNA except the terminal base pairs. CysMeTANDEM binds to and affects the structure of the DNA in a manner similar to that observed in complexes of the CpG-specific quinoxaline antibiotics triostin A and echinomycin with DNA [Ughetto, G., Wang, A. H.-J., Quigley, G. J., van der Marel, G. A., van Boom, J. H., & Rich, A. (1985) Nucleic Acids Res. 13, 2305-2323; Wang, A. H.-J., Ughetto G., Quigley, G. J., Hakoshima, T., van der Marel, G. A., van Boom, J. H., & Rich, A. (1984) Science 225, 1115-1121; Wang, A. H.-J., Ughetto, G., Quigley, G. J., & Rich, A. (1986) J. Biomol. Struct. Dyn. 4, 319-342]. The two quinoxaline rings bisintercalate on either side of the two central T.A base pairs and the peptide ring lies in the minor groove. The central A.T base pairs of the complex are underwound (average helical twist angle of approximately -10 degrees) and buckle inward by approximately 20 degrees. There are intermolecular hydrogen bonds between each of the Ala NH and the AN3 protons of the TpA binding site, analogous to those observed between Ala NH and GN3 in the crystal structures of the CpG-specific complexes of echinomycin and triostin A with DNA. However, the structure of the peptide ring of CysMeTANDEM in the complex differs from that of echinomycin and triostin A.(ABSTRACT TRUNCATED AT 400 WORDS)