Studies of the B-Z transition of DNA: The temperature dependence of the free-energy difference, the composition of the counterion sheath in mixed salt, and the preparation of a sample of the 5'-d[T-(m(5) C-G)12 -T] duplex in pure B-DNA or Z-DNA form.

It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution. Here, we analyze the non-electrostatic component of the free-energy difference between B-DNA and Z-DNA. We also compute the composition of the counterion sheath in a wide range of mixed-salt solutions and of temperatures: in contrast with the large difference of composition between the B-DNA and Z-DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed-salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small-Angle X-ray Scattering and complemented by (31) P NMR. With due consideration for the kinetics of the B-Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5'-d[T-(m(5) C-G)12 -T] 26-mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson-Boltzmann equation is described in detail. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 369-384, 2016.

Innovative integrated system for real-time measurement of hybridization and melting on standard format microarrays.

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.

Sensitive CEST agents based on nucleic acid imino proton exchange: detection of poly(rU) and of a dendrimer-poly(rU) model for nucleic acid delivery and pharmacology.

It is shown that the exchange properties of the imino and hydroxyl protons of polyuridilic acid (poly(rU)) allow use of this compound as a chemical-exchange saturation transfer (CEST) contrast agent. A proton/proton sensitivity enhancement factor of over 5000 per imino proton allowed the detection of a few micromolar of polymer (2000 uridine units; 644 kD) with a 50% change in the water signal. The enhancement factor would increase further at even lower concentrations, opening up the submicromolar range. When poly(rU) was complexed to a dendrimer carrying 250 positive charges, the stoichiometry was approximately one RNA for 10 dendrimers. The sensitivity enhancement was reduced but remained large (2300/imino proton), bringing enhanced CEST visibility to the dendrimers. The net charge of the complex was positive, suggesting that the complexed dendrimers would still interact with cell membranes, and that the RNA-dendrimer complex could provide a model for a gene delivery system with good CEST visibility.

The Doppler effect in NMR spectroscopy.

An NMR sample may be subject to motions, such as those due to sample spinning or to liquid flow. Is the spectrum of such a sample affected by the Doppler effect? The question arises because, instrumental dimensions being much shorter than the wavelength, it is the near-field of the precessing magnetic moment which couples to the receiver coil, rather than the radiated far-field. We expand the near-field into plane propagating waves. For each such wave there is another one with the same amplitude, propagating in the opposite direction. The Doppler shifts are therefore equal and opposite. In the model case of a small fluid sample moving with constant velocity, this leads to a distribution of Doppler shifts which is symmetrical with respect to the unshifted frequency: there is no net spectral shift. We examine the possibility of observing the Doppler distribution in this case. We also consider the case of thermal motion of a gas. We draw attention to the resolved Doppler splitting of molecular rotational transitions in a supersonic burst as observed in a microwave resonator. We also mention briefly the Doppler effect in molecular beam spectroscopy.

Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand.

As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC). A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band. The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment. Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p. These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers. The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded. We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.