Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA

Identification of Allele-Specific RNAi Effectors Targeting Genetic Forms of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5–10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a ‘walk-through’ series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the α-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5′RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future

Adding to Hans Kuhn's thesis on the emergence of the genetic apparatus of the darwinian advantage to be neither too soluble, nor too insoluble, neither too solid, nor completely liquid

International audienceA scenario of the origin of the genetic apparatus is described where the surface and physico-chemical properties of lipid bilayers and multilayers of vesicles play a crucial role. Peptides, nucleic acids and lipids are 'collaborating' to bring about a first successful genetic apparatus. Lipidic vesicles acquire new properties through hosting nucleic acids that are transiently but covalently linked to lipophilic peptides. These peptides anchor the associated nucleic acids into the surface of lipidic vesicles. In the interior of such vesicles, within the lipidic bilayer, peptidyl transfers occur that are reminiscent of modern-day ribosomal peptidyl transfer reactions. One can expect that the growing peptides eventually acquire, stepwise and essentially arbitrarily, new functions different from anchoring nucleic acids, such as specific aminoacylation of nucleic acids, template-assisted nucleic acid synthesis, nucleotide deoxygenation and fatty acid synthesis

The Beginning of Systems Chemistry

Systems Chemistry has its roots in the research on the autocatalytic self-replication of biological macromolecules, first of all of synthetic deoxyribonucleic acids. A personal tour through the early works of the founder of Systems Chemistry, and of his first followers, recalls what’s most important in this new era of chemistry: the growth and evolution of compartmented macromolecular populations, when provided with “food„ and “fuel„ and disposed of “waste„

Omne Vivum ex vivo ... Omne

International audience“What I cannot create, I do not understand.” Richard Feynman’s bon mot seems almost tailored for experimental systems chemists to realise an important step in the chemical sciences: the creation of living synthetic cells from the entirely inanimate. The underlying idea needs to be simple so the system can develop naturally. This proposal aims at the realisation, viz. finding sets of experimentally feasible initial conditions, exploring varied compositions and analysing their outcomes, of a fully synthetic chemical micro-compartmented and evolvable macromolecular system being fed with monomers and small molecular weight, high energy compounds, to keep the system permanently out of thermodynamic equilibrium and let it self-evolve, thus gaining: 1) import-export control of macromolecules across the compartment membranes; 2) food-dependent increase in macromolecular size, i.e., polymer length inside the compartments; 3) sustained production of new macromolecules through the establishment of a (or several) de novo genetic code(s); leading to 4) the emergence of replicating macromolecular populations; and 5) the emergence of self-evolved synthetic living cells