Biomedical subjects

Arthur Pardi

Publications and source records attributed to Arthur Pardi.

10 recordsLinked to original sources

PubMed2006

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker.

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Base Sequence↗

PubMed2005

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165.

Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2'fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF(165) aptamer binds specifically to the VEGF(165) isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF(165)-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF(165) for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF(164)-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF(165)-aptamer complex.

Aptamers, Nucleotide↗

PubMed2005

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.

Base Sequence↗

PubMed2005

Comparison of the global structure and dynamics of native and unmodified tRNAval.

The effects of post-transcriptional modifications on the structure and dynamics of Escherichia coli tRNA(val) were studied by NMR spectroscopy. NMR chemical shift data and residual dipolar couplings were used to show that the local secondary and tertiary structures are very similar in native and unmodified tRNA(val). Rigid body restrained molecular dynamics calculations showed that the global structure of tRNA is unchanged by the post-transcriptional modifications. Deuterium exchange NMR experiments were used to probe the dynamics and flexibility of native and unmodified tRNA(val). A similar set of slowly exchanging (t(1/2) > 3 min) imino protons were observed in both tRNAs, but the rates of exchange for the slowest exchanging imino protons were approximately 20 times faster in unmodified than in native tRNA. These results demonstrate that the dynamics and flexibility of tRNA(val), but not the local or global structure, are significantly affected by post-transcriptional modifications.

Base Sequence↗

PubMed2005

NMR methods for studying the structure and dynamics of RNA.

Proper functioning of RNAs requires the formation of complex three-dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope-labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen-bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long-range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating-frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

Hydrogen Bonding↗

PubMed2005

Resolution-enhanced base-type-edited HCN experiment for RNA.

New base-type-edited transverse-relaxation optimized CT-HCN(C) experiments are presented that yield intra-base and sugar-to-base correlations for 13C-15N labeled RNA. High spectral resolution in the 13C and 15N dimensions is achieved by constant time (CT) frequency editing. A spectral editing filter applied during the CT 15N labeling period separates the correlation peaks arising from G/U and A/C nucleotide bases. This provides the increased spectral resolution required to unambiguously connect the 1H and 13C resonances in sugar and base moieties of RNA nucleotides. In addition, the experiment allows base type identification for each residue, and therefore presents an attractive spectroscopic alternative to nucleotide-specific isotope labeling. Application to a 33-nucleotide RNA aptamer demonstrates the performance of the new pulse scheme.

Carbon Isotopes↗

PubMed2004

Fast cleavage kinetics of a natural hammerhead ribozyme.

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.

Animals↗

PubMed2003

Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex.

The helical regions of RNA are generally very stable, but the single-stranded and loop regions often exist as an ensemble of conformations in solution. The theophylline-binding RNA aptamer forms a very stable structure when bound to the bronchodilator theophylline, but the theophylline binding site is not stably formed in the absence of ligand. The kinetics for theophylline binding were measured here by stopped-flow fluorescence spectroscopy to probe the mechanism for theophylline binding in this RNA aptamer. The kinetic studies showed that formation of the RNA-theophylline complex is over 1000 times slower than a diffusion-controlled rate, and the high affinity of the RNA-theophylline complex arises primarily from a slow dissociation rate for the complex. A theophylline-independent rate was observed for formation of the theophylline-RNA complex at high theophylline concentration, indicating that a conformational change in the RNA is the rate-limiting step in complex formation under these conditions. The RNA-theophylline complex requires divalent metal ions, such as Mg2+, to form a high-affinity complex, and there is a greater than 10000-fold reduction in affinity for theophylline in the absence of Mg2+. This decrease in binding affinity in the absence of Mg2+ results primarily from an increased dissociation rate for the complex. The implications of an ensemble of conformations in the free state of this theophylline-binding RNA are discussed and compared with mechanisms for formation of protein-ligand complexes.

2-Aminopurine↗

PubMed2003

Refined solution structure of the iron-responsive element RNA using residual dipolar couplings.

The iron-responsive element (IRE) is a 30nt RNA motif located in the non-coding regions of mRNAs of proteins involved in iron regulation. In humans, the IRE plays a direct role in the control of iron levels by post-transcriptional regulation of the ferritin and transferrin receptor proteins through highly specific recognition by IRE-binding proteins. The IRE fold is representative of many RNA motifs that contain helical domains separated by a bulge or internal loop. The global structures of such extended multi-domain RNAs are not well defined by conventional NMR-distance and torsion angle structural restraints. Residual dipolar couplings (RDCs) are employed here to better define the global structure of the IRE RNA in solution. RDCs contain valuable long-range structural information that compliments the short-range structural data derived from standard NOE-distance and torsion angle restraints. Several approaches for estimating alignment tensor parameters and incorporating RDCs into RNA structure determinations are compared. Both the local and global structure of the IRE are improved significantly by refinement with RDCs. These RDC refinements provide insight on the conformational dynamics of the IRE. These studies highlight some issues that need to be addressed when incorporating RDCs in solution structure determinations of nucleic acids. The approach used here should prove valuable for structure determinations of various multi-domain systems.

Humans↗

PubMed2002

The global conformation of the hammerhead ribozyme determined using residual dipolar couplings.

The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.

Catalytic Domain↗