Synthetic analogue approach to metallobleomycins: syntheses, structure and properties of mononuclear and tetranuclear gallium(III) complexes of a ligand that resembles the metal-binding site of bleomycin

As part of our interest into the bioinorganic chemistry of gallium, gallium(III) complexes of the peptide ligand N-(2-(4-imidazolyl)ethyl)pyridine-2-carboxamide (pypepH2) resembling a fragment of the metal-binding domain of bleomycins (BLMs), have been isolated. Reaction of pypepH2 with (Et4N)[GaCl4] and Ga(acac)3 [acac- is the acetylacetonate(-1) ion] affords the mononuclear complex [Ga(pypepH)2]Cl.2H2O (1) and the tetranuclear complex [Ga4(acac)4(pypep)4].4.4H2O (2), respectively. Both complexes were characterized by single-crystal X-ray crystallography, IR spectroscopy and thermal decomposition data. The pypepH- ion in 1 behaves as a N(pyridyl), N(deprotonated amide), N(pyridine-type imidazole) chelating ligand. The doubly deprotonated pypep2- ion in 2 behaves as a N(pyridyl), N(deprotonated amide), N(imidazolate), N'(imidazolate) mu2 ligand and binds to one Ga(III) atom at its pyridyl, amide and one of the imidazolate nitrogens, and to a second metal ion at the other imidazolate nitrogen; a chelating acac- ligand completes six coordination at each Ga(III) centre. The IR data are discussed in terms of the nature of bonding and known structures. The 1H NMR spectrum of 1 suggests that the cation of the complex maintains its integrity in dimethylsulfoxide (DMSO) solution. Complexes 1 and 2 are the first synthetic analogues of metallobleomycins with gallium(III).     

Rational siRNA design for RNA interference

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.     

Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

It has been shown that the activity of the hammerhead ribozyme at microM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the microM range of magnesium ion concentration. Disruption of the loop-loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions.     

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 microM MgCl2. The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80 degrees C in 10 mM MgCl2 and at 70 degrees C in 2 mM MgCl2. A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl2, including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.     

<Emphasis Type="Italic">Thermus thermophilus</Emphasis> genome analysis: benefits and implications

The genome sequence analysis of Thermus thermophilus HB27, a microorganism with high biotechnological potential, has recently been published. In that report, the chromosomal and the megaplasmid sequence were compared to those of other organisms and discussed on the basis of their physiological and metabolic features. Out of the 2,218 putative genes identified through the large genome sequencing project, a significant number has potential interest for biotechnology. The present communication will discuss the accumulating information on molecules participating in fundamental biological processes or having potential biotechnological importance.     

Synthesis of chroman analogues of lipoic acid and evaluation of their activity against reperfusion arrhythmias

Novel hybrids of lipoic acid and trolox connected through triamine spacers as well as analogues in which the lipoic acid was attached at different positions of the chroman moiety of vitamin E through an amide bond, were synthesized and exhibited strong inhibition of the microsomal lipid peroxidation. Moreover, the new molecules, at 1 microM concentration, reduced reperfusion arrhythmias and MDA content on isolated rat heart preparations, with the 2- and 5-subtituted chromans possessing the better cardioprotective activity.     

MMDB: Entrez's 3D-structure database

Three-dimensional structures are now known within most protein families and it is likely, when searching a sequence database, that one will identify a homolog of known structure. The goal of Entrez's 3D-structure database is to make structure information and the functional annotation it can provide easily accessible to molecular biologists. To this end, Entrez's search engine provides several powerful features: (i) links between databases, for example between a protein's sequence and structure; (ii) pre-computed sequence and structure neighbors; and (iii) structure and sequence/structure alignment visualization. Here, we focus on a new feature of Entrez's Molecular Modeling Database (MMDB): Graphical summaries of the biological annotation available for each 3D structure, based on the results of automated comparative analysis. MMDB is available at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html.     

A new dose-finding design for bivariate outcomes

For some drugs, toxicity events lead to early termination of treatment before a therapeutic response is observed. That is, there are three possible outcomes: toxicity (therapeutic response unknown), therapeutic response without toxicity, and no response with no toxicity. The optimal dose is the dose that maximizes the probability of the joint event, response, and no toxicity. The optimal safe dose is the dose, from among the doses with toxicity rate less than the maximum tolerable level, that maximizes the probability of response and no toxicity. We present a new sequential design to maximize the number of subjects assigned in the neighborhood of the optimal safe dose in a dose-finding trial with two outcomes.