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J Feigon

Publications and source records attributed to J Feigon.

At least 19 recordsLinked to original sources
PubMed1997

Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding.

The solution structure of the ATP-binding RNA aptamer has recently been determined by NMR spectroscopy. The three-dimensional fold of the molecule is determined to a large extent by stacking and hydrogen bond interactions. In the course of the structure determination it was discovered that several highly conserved nucleotides in the binding pocket can be substituted while retaining binding under NMR conditions. These surprising findings allow a closer look at the interactions that determine stability and specificity of the aptamer as well as local structural features of the molecule. The binding properties of ATP binder mutants and modified ligand molecules are explored using NMR spectroscopy, column binding studies and molecular modeling. We present additional evidence and new insights regarding the network of hydrogen bonds that defines the structure and determines stability and specificity of the aptamer.

Adenosine Monophosphate↗
PubMed1997

Solution structure of the conserved 16 S-like ribosomal RNA UGAA tetraloop.

The solution structure of the highly conserved UGAA tetraloop found at the 3' end of eukaryotic 16 S-like ribosomal RNA has been solved by nuclear magnetic resonance spectroscopy in the form of the 12 nucleotide hairpin 5'-GGUG[UGAA]CACC. The UGAA tetraloop displays a novel fold. The backbone turn occurs between the G and the third A in the loop, with the U and G in a 5' stack and the As in a 3' stacking arrangement. The loop is closed by a U-A mismatch in which the O2, 2'OH, and O4' groups of the U are within hydrogen bonding distance of the amino group of the A. The tetraloop does not make a uridine-turn, even though its sequence is identical to a U-turn found within the anticodon loop of tRNA(Phe). The hydrogen bonding pattern in the tetraloop provides insight into the function of base modifications found in vivo within this portion of 16 S-like rRNA.

Hydrogen Bonding↗
PubMed1997

Solution structure of an intramolecular DNA triplex containing an N7-glycosylated guanine which mimics a protonated cytosine.

The three-dimensional structure of a pyrimidine-purine-pyrimidine DNA triplex containing an N7-glycosylated guanine (7G) in the third strand has been determined by NMR spectroscopy, restrained molecular dynamics calculations, and complete relaxation matrix refinement. Glycosylation of the guanine at the N7 position permits it to adopt a conformation such that the Hoogsteen face of the base mimics the arrangement of hydrogen bond donors seen in protonated cytosine. The NMR data confirm the previously proposed hydrogen bonding scheme of the 7G x G x C triplet. The three-dimensional structure of the triplex accommodates the 7G with less distortion of the phosphodiester backbone than would be required for an N9-glycosylated guanine in the same sequence position, but some changes in the positions of the phosphodiester backbone are present compared to a C+ x G x C triplet. The structure provides a rationale for the observations that 7G binds to Watson-Crick G x C base pairs with higher specificity and affinity than guanine, but with a lower stability at pH 5.2 than would be provided by a canonical C+ x G x C triplet.

Base Composition↗
PubMed1997

Assignment methodology for larger RNA oligonucleotides: application to an ATP-binding RNA aptamer.

The use of uniform 13C, 15N labeling in the NMR spectroscopic study of RNA structures has greatly facilitated the assignment process in small RNA oligonucleotides. For ribose spin system assignments, exploitation of these labels has followed previously developed methods for the study of proteins. However, for sequential assignment of the exchangeable and nonexchangeable protons of the nucleotides, it has been necessary to develop a variety of new NMR experiments. Even these are of limited utility in the unambiguous assignment of larger RNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei. These problems can largely be overcome by the additional use of basetype selectively 13C, 15N-labeled RNA in combination with a judicious use of related RNAs with base substitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNA aptamer in complex with AMP. Complete sequential 1H assignments, as well as the majority of 13C and 15N assignments, were obtained.

Adenosine Triphosphate↗
PubMed1996

Structural change in Rev responsive element RNA of HIV-1 on binding Rev peptide.

The HIV-1 Rev responsive element (RRE) high-affinity binding site was studied by homonuclear and heteronuclear NMR. Two Rev binding element (RBE) RNA oligonucleotides were used as model systems in this study: RBE3, which contains the wild-type Rev high-affinity binding site, and RBE3-A which is identical except for the deletion of a bulged A. The temperature dependence of the two-dimensional spectra of the free RNAs indicates that at lower temperatures more than one conformation is present. However, at higher temperatures a single conformation predominates. Model structures of RBE3 and RBE3-A as well as the RBE3-A complexed with a peptide derived from the RNA binding domain of HIV-1 Rev, were calculated using NMR-derived restraints. The Rev high-affinity binding site of the HIV-1 RRE contains a structured internal loop with two purine-purine base-pairs and an extrahelical U. Comparison of the free and bound RNA structures reveals that upon peptide binding there is a distinct change in the backbone at G24, which is involved in a G-G base-pair. In the free RNA, G24 is in the syn conformation, and the backbone is in a relatively normal configuration, antiparallel to the other strand. In the bound RNA, the backbone at G24 has flipped over so that it is parallel to the other strand. G24 in the bound RNA still forms a base-pair with G6, but is now in the anti conformation.

Amino Acid Sequence↗
PubMed1996

The selectivity for K+ versus Na+ in DNA quadruplexes is dominated by relative free energies of hydration: a thermodynamic analysis by 1H NMR.

We have studied the competition between Na+ and K+ for coordination by G quartets using the oligonucleotide d(G3T4G3) as a model system. d(G3T4G3) forms a dimeric foldback structure containing three G quartets in the presence of either NaCl or KCl. Proton chemical shifts, which are particular to the species of coordinated ion, have been used to monitor the conversion between the sodium and potassium forms under equilibrium conditions. Analysis of titration experiments indicates that at least two K+ are coordinated by the three quartets of the dimeric molecule, and perfect fits of the data are obtained for two Na+ being displaced by two K+. Our results also indicate that the conversion of [d(G3T4G3)]2 from the sodium to the potassium form is associated with a net free energy change (delta G degrees) of -1.7 +/- 0.15 kcal/mol. It has long been suggested that the greater thermal stability of DNA quadruplex structures in the presence of K+ is primarily a result of the optimal fit of this ion in the coordination sites formed by G quartets. However, a consideration of the relatively small change in free energy associated with the conversion from the sodium to the potassium form and the relatively large difference between the free energy of hydration for Na+ and K+ indicates that this cannot be correct. Rather, the preferred coordination of K+ over Na+ is actually driven by the greater energetic cost of Na+ dehydration with respect to K+ dehydration.

DNA↗
PubMed1996

Solution structure of a pyrimidine-purine-pyrimidine triplex containing the sequence-specific intercalating non-natural base D3.

We have used NMR spectroscopy to study a pyrimidine-purine-pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3), in the third strand. The D3 base has been previously shown to specifically recognize T-A and C-G base-pairs via intercalation on the 3' side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D3 base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D3 base stack on the bases of all three strands and mimic a triplet. The conformation of the D3 base and its sequence specificity are discussed.

Base Sequence↗
PubMed1996

Reconciliation of the X-ray and NMR structures of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG).

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) is one of a family of DNA oligonucleotides that were identified by in vitro selection to bind specifically and with high affinity to thrombin. Two groups independently determined the tertiary structure in solution by NMR and at about the same time, the X-ray crystal structure of the aptamer in complex with thrombin was reported. In all cases, the thrombin-binding aptamer was found to fold into a structure containing two planar guanine quartets as its core. The NMR and crystal structures, however, have fundamentally different folding patterns owing to differences in the way these central bases are connected. We discuss the distinctions between the refined crystal and solution structures and show that the NMR model is consistent with the X-ray diffraction data.

Base Sequence↗
PubMed1996

Through-bond correlation of imino and aromatic resonances in 13C-, 15N-labeled RNA via heteronuclear TOCSY.

Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly 15N-, 13C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), 51 as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.

Base Composition↗
PubMed1996

Aptamer structures from A to zeta.

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.

Adenosine Triphosphate↗
PubMed1996

Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes.

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.

Amino Acid Sequence↗
PubMed1996

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

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.

Adenosine Monophosphate↗
PubMed1995

Solution structures of unimolecular quadruplexes formed by oligonucleotides containing Oxytricha telomere repeats.

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.

Animals↗
PubMed1995

High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus.

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.

Antiviral Agents↗
PubMed1994

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

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.

Anti-Bacterial Agents↗