Polar nanoregions in Pb(Mg<Subscript>1/3</Subscript>Nb<Subscript>2/3</Subscript>)O<Subscript>3</Subscript> (PMN): insights from a supercell approach

We report construction of a model of polar nanoregions in the PMN relaxor ferroelectric based on first-principles lattice dynamics for chemically ordered supercells [S.A. Prosandeev et al., Phys. Rev. B 70, 134110 (2004)], combined with invariance under permutations and dipole-dipole interaction as a source supporting randomly oriented residual polarization. Representative analytical estimates of polar nanore-gion — supercell mapping reproduce both nonzero local and zero macroscopic polarization of the structure, as well as the temperature change of the supercell anisotropy at cooling and field cooling.     

Deterministic and statistical methods for reconstructing multidimensional NMR spectra

Reconstruction of an image from a set of projections is a well-established science, successfully exploited in X-ray tomography and magnetic resonance imaging. This principle has been adapted to generate multidimensional NMR spectra, with the key difference that, instead of continuous density functions, high-resolution NMR spectra comprise discrete features, relatively sparsely distributed in space. For this reason, a reliable reconstruction can be made from a small number of projections. This speeds the measurements by orders of magnitude compared to the traditional methodology, which explores all evolution space on a Cartesian grid, one step at a time. Speed is of crucial importance for structural investigations of biomolecules such as proteins and for the investigation of time-dependent phenomena. Whereas the recording of a suitable set of projections is a straightforward process, the reconstruction stage can be more problematic. Several practical reconstruction schemes are explored. The deterministic methods-additive back-projection and the lowest-value algorithm-derive the multidimensional spectrum directly from the experimental projections. The statistical search methods include iterative least-squares fitting, maximum entropy, and model-fitting schemes based on Bayesian analysis, particularly the reversible-jump Markov chain Monte Carlo procedure. These competing reconstruction schemes are tested on a set of six projections derived from the three-dimensional 700-MHz HNCO spectrum of a 187-residue protein (HasA) and compared in terms of reliability, absence of artifacts, sensitivity to noise, and speed of computation.     

Hyperdimensional NMR spectroscopy

Hyperdimensional NMR describes the multiple interactions between a large number, N, of coupled nuclear spins. We show how to construct a hypothetical N-dimensional spectrum from a limited set of low-dimensional measurements, exploiting the concept of projection-reconstruction. In the process, many correlation spectra are obtained that were not investigated directly. As an illustration, the hypothetical ten-dimensional spectrum of a small protein (agitoxin) has been reconstructed, and a large number of pairwise correlation spectra have been recorded.     

Total synthesis of 3',5'-C-branched nucleosides

[reaction: see text] A novel total synthesis of 3',5'-C-branched uridine azido acid has been accomplished using stereoselective aldehyde alkynylation, Ireland-Claisen rearrangement, and iodolactonization as the key reactions. Compared to traditional routes that start from carbohydrates, the present methodology is more efficient, flexible for future optimization, and provides access to both enantiomers of the products. Because the key chemistry does not involve the 3'- and 5'-C substituents, our route is a general approach to 3',5'-C alkyl nucleoside analogues.     

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.     

Synthetic,Structural, and RNA Binding Studies on 2-Aminopyridine-Modified Triplex-Forming Peptide Nucleic Acids

The development of new RNA-binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex-forming peptide nucleic acids (PNAs) by further increasing the pK_a of 2-aminopyridine ( M ). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron-donating 4-methoxy substituent resulted in slight destabilization of the PNA–dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M -modified PNA–dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4-methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen-bonding interactions that might be responsible for the high affinity and unusual RNA-binding preference of PNAs.     

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3′-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5′-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.     

Adiabatic Mixing in the Liquid State

Adiabatic spin inversion has been used in the liquid state very efficiently for decoupling purposes. Here we show that it can also be adapted for spin mixing experiments, such as the TOCSY and clean TOCSY experiment, and is superior to previously employed mixing sequences. The main advantage of adiabatic mixing sequences over the conventional mixing schemes used in liquid state experiments is an extremely low sensitivity to RF field inhomogeneity and miscalibration of theB₁field strength. The method is evaluated experimentally by comparing results obtained with different mixing schemes in the basic 2D TOCSY experiment. In addition to higher reliability, adiabatic mixing provides a sensitivity improvement of ca. 20% as compared to conventional mixing schemes. This is explained by higher signal losses due to RF inhomogeneity in the experiments employing traditional mixing schemes. More significant sensitivity improvements can be expected in situations where RF homogeneity is traditionally poor, for example, in large volume probes and magnetic resonance imaging experiments.     

NMR at 900 MHz

An important factor in the development of solution state NMR has always been th e ability to produce stable and homogeneous magnetic fields. As higher and higher field strengths are reached the pressure is growing on manufacturers to produce NMR systems with greatly improved spectral resolution and signal to noise ratio. The introduction of the Varian 900 MHz INOVA system in August 2000 featuring Oxford Instruments 21.1 T magnet represents the latest pioneering development in NMR technology.