Nuclease resistant hammerhead motif: from '5-ribo' to '3-ribo' model

Previously developed '5-ribo' nuclease stabilized hammerhead motif was further refined by systematic incorporation of 1-(beta-D-xylofuranosyl) adenine (xA) and 1-(beta-D-xylofuranosyl) guanine (xG) in the place of conserved ribopurine residues of the catalytic core. Modified ribozymes substituted with xA at positions A15.1 and A6 demonstrated catalytic activity close to the parent stabilized ribozyme. Analogous guanosine substitutions at positions G5, G8, and G12 substantially lowered catalytic rates.     

Effect of 3-5-3'triiodothyronine treatment on cardiac action potential of streptozotocin-induced diabetic rat

Electrical activity of rat atrium of streptozotocin-diabetic and control rats was compared. (i) As occurs in the ventricle, diabetes lengthens the cardiac atrial action potential. (ii) Treatment by T3 of diabetic animals decreases action potential duration to normal values and causes partial recovery in plateau decay during the late phase of repolarization. (iii) T3 treatment however, does not completely normalized the action potential of the diabetic rat atrium, which remains abnormal during the early phase of repolarization. These results demonstrate that some defects in membrane mechanisms involved in the early phase of action potential repolarization are attributable solely to diabetes. The possible nature of these mechanisms is discussed.     

Synthesis of 5′-C-Methyl-D-allo- & L-Talo-ribonucleoside 3′-O-Phosphoramidites & Their Incorporation into Hammerhead Ribozymes

Abstract

5′-C-Methyl-D-allo & L-talo-ribonucleoside 3′-O-phosphoramidites were prepared from L-rhamnose in 13 and 15 steps respectively. Incorporation of L-talo residues in the hammerhead ribozyme and the resulting activity and stability of the modified ribozymes is described.     

Synthesis and enzymatic deprotection of fully protected 2'-5' oligoadenylates (2-5A): towards a prodrug strategy for short 2-5A

Fully protected pA2'p5'A2'p5'A trimers 1a and 1b have been prepared as prodrug candidates for a short 2'-5' oligoadenylate, 2-5A, and its 3'-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH?7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3'-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2',5'→3',5' phosphate migration and release of adenosine as side reactions.     

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.     

Structure-function relationships in the hammerhead ribozyme probed by base rescue.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.     

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.     

The development of a monoclonal antibody specific for a 2(')-C-allyl modification of uridine,and its use in the localization of ribozymes in vivo

Ribozymes are catalytically active RNA molecules that cleave other RNA molecules in a sequence-specific fashion, with significant turnover. The successful design and synthesis of ribozymes with modifications to increase their stability in biological fluids, while maintaining catalytic activity, has been instrumental in moving this technology from the laboratory into clinical trials. With the entry of ribozymes into the clinical setting, the need has arisen for reagents and/or assays to detect these drugs in tissues. We have developed a monoclonal antibody to the 2(')-deoxy-2(')-C-allyl uridine modification present in our synthetic hammerhead ribozymes. The monoclonal antibody, termed CA1USR, is a murine IgG1(k), whose epitope appears to involve both the 2(')-C-allyl modification, and the uridine base. Use of CA1USR for immunohistochemical detection of ribozymes in the tissues of mice which were administered two structurally different ribozymes has demonstrated its utility as a reagent for in vivo localization of ribozymes containing the 2(')-C-allyl uridine modification.     

Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface

The MS2 coat protein binds specifically to an RNA hairpin formed within the viral genome. By soaking different RNA fragments into crystals of MS2 coat protein capsids it is possible to determine the X-ray structure of the RNA–protein complexes formed. Here we present the structure to 2.85 Å resolution of a complex between a chemically modified RNA hairpin variant and the MS2 coat protein. This RNA variant has a substitution at the –5 base position, which has been shown previously to be pyrimidine-specific and is a uracil in the wild-type RNA. The modified RNA hairpin contains a pyridin-4-one base (4one) at this position that lacks the exocyclic 2-oxygen eliminating the possibility of forming a hydrogen bond to asparagine A87 in the protein. The 4one complex structure shows an unprecedented major conformational change in the loop region of the RNA, whereas there is almost no change in the conformation of the protein.     

Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines

Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.