Imino proton exchange rates imply an induced-fit binding mechanism for the VEGF165-targeting aptamer, Macugen.

The 2'-fluoro/2'-O-methyl modified RNA aptamer Macugen is a potent inhibitor of the angiogenic regulatory protein, VEGF165. Macugen binds with high affinity to the heparin-binding domain (HBD) of VEGF165. Hydrogen exchange rates of the imino protons were measured for free Macugen and Macugen bound to the HBD or full-length VEGF to better understand the mechanism for high affinity binding. The results here show that the internal loop and hairpin loop of Macugen are highly dynamic in the free state and are greatly stabilized and/or protected from solvent upon protein binding.

Efficient ligation of the Schistosoma hammerhead ribozyme.

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

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.

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.

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.