Real-Time Detection of a Self-Replicating RNA Enzyme.

A system was developed to detect the self-replication of an RNA enzyme in real time. The enzyme is an RNA ligase that undergoes exponential amplification at a constant temperature and can be made to operate in a ligand-dependent manner. The real-time system is based on a fluorimetric readout that directly couples the ligation event to an increase in florescence signal that can be monitored using standard instrumentation.

An L-RNA Aptamer that Binds and Inhibits RNase.

L-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates.

Ligand-dependent exponential amplification of self-replicating RNA enzymes.

A general analytical method for the detection of target ligands has been developed, based on a special class of self-replicating aptazymes. These "autocatalytic aptazymes" are generated by linking an aptamer domain to the catalytic domain of a self-replicating RNA enzyme. Ligand-dependent self-replication of RNA proceeds in a self-sustained manner, undergoing exponential amplification at a constant temperature without the assistance of any proteins or other biological materials.

Ligand-dependent exponential amplification of a self-replicating L-RNA enzyme.

A nuclease-resistant RNA enzyme, constructed entirely from L-ribonucleotides, was shown to undergo ligand-dependent, self-sustained replication with exponential growth. The catalytic motif is based on a previously described RNA ligase that can undergo either self- or cross-replication but had been limited in its application to ligand sensing due to its susceptibility to degradation by ribonucleases. The self-replicating RNA enzyme and its RNA substrates were prepared synthetically from either D- or L-nucleoside phosphoramidites.

Modulating heme redox potential through protein-induced porphyrin distortion.

Hemoproteins are ubiquitous in biology and are commonly involved in critical processes such as electron transfer, oxidative phosphorylation, and signal transduction. Both the protein environment and the heme cofactor contribute to generate the range of chemical properties needed for these diverse functions. Using the heme nitric oxide/oxygen binding (H-NOX) protein from the thermophilic bacterium Thermoanaerobacter tengcongensis, we have shown that heme electronic properties can be modulated by porphyrin distortion within the same protein scaffold without changing the heme ligation state or heme environment.

Structure and properties of a bis-histidyl ligated globin from Caenorhabditis elegans.

Globins are heme-containing proteins that are best known for their roles in oxygen (O(2)) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes were recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O(2) sensing behavior.

Ru-porphyrin protein scaffolds for sensing O2.

Hemoprotein-based scaffolds containing phosphorescent ruthenium(II) CO mesoporphyrin IX (RuMP) are reported here for oxygen (O(2)) sensing in biological contexts. RuMP was incorporated into the protein scaffolds during protein expression utilizing a novel method that we have described previously. A high-resolution (2.

Structural insights into the molecular mechanism of H-NOX activation.

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron-histidine (Fe-His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H-NOX (Heme-Nitric oxide/OXygen) binding domains.

Probing the function of heme distortion in the H-NOX family.

Hemoproteins carry out diverse functions utilizing a wide range of chemical reactivity while employing the same heme prosthetic group. It is clear from high-resolution crystal structures and biochemical studies that protein-bound hemes are not planar and adopt diverse conformations. The crystal structure of an H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) contains the most distorted heme reported to date.

Crystallization and initial X-ray analysis of phenoxazinone synthase from Streptomyces antibioticus.

Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown.