Synthesis and nucleic acid binding studies of novel pyrrolidinyl PNA carrying an N-amino-N-methylglycine spacer

Two novel pyrrolidinyl peptide nucleic acids comprising alternating sequences of thymine-modified d- or l-proline and an N-amino-N-methylglycine spacer were synthesized using solid-phase methodology. UV and CD titrations together with a gel-binding shift assay revealed that neither of the homothymine PNA decamers bind to their complementary DNA or RNA. This was considered to be due to an unfavorable secondary structure which could not be alleviated by the presence of the positively charged protonated amine in the PNA backbone.     

Comparison of several curve resolution methods for drug impurity profiling using high-performance liquid chromatography with diode array detection

The performance of five curve resolution methods was compared systematically for the identification and quantification of impurities in drug impurity profiling. These methods are alternating least-squares (ALS) with either random or iterative key-set factor analysis (IKSFA) initialisation, iterative target transformation factor analysis (ITTFA), evolving factor analysis (EFA), and heuristic evolving latent projections (HELP). Real and simulated high-performance liquid chromatography diode array detection (HPLC-DAD) data were obtained for drug mixtures containing one main compound and two impurities. The elution order of the main compound and the impurities was varied. Furthermore, resolutions were varied from 0.56 to 3.36 and impurity levels from 30% down to 0.1%. For simulated data, ALS with IKSFA initialisation and HELP perform better than ITTFA and EFA, which perform better than ALS with random initialisation. ITTFA works better than EFA for almost completely separated data, while the opposite is true for moderately or strongly overlapping data. Only ALS with IKSFA initialisation and HELP were found to resolve the required 0.1% level for moderately overlapping data. For real data, comparison of the methods provides similar results. ITTFA performs clearly better than EFA. However, none of the curve resolution methods can identify or quantify impurities at the required 0.1% level. The results for real data are worse than for simulated data because of heteroscedasticity, nonlinearity, and the acquisition resolution of the A/D-converter.     

Multiple ionization and fragmentation of the DNA base thymine by interaction with C q+ ions

Ionization and fragmentation of the DNA base thymine upon interaction with keV C^q+ ions (q = 1 - 6) has been studied. By means of time-of-flight spectrometry of two or more thymine fragments in coincidence with an ejected electron we could investigate particular dissociation channels by means of their associated kinetic-energy-release. The fragmentation dynamics are strongly influenced by the C^q+ charge state: for low q values mainly fragmentation due to direct collisions is observed. With increasing q, electron capture becomes more important. For larger q we could identify several Coulomb explosion channels, leading to very energetic fragments.     

Insights into nucleic acid reactivity through gas-phase experimental and computational studies

Accurate measurements of the acidities and basicities of nucleic bases and nucleic base derivatives is essential for understanding issues of fundamental importance in biological systems. Hydrogen bonding modulates recognition of DNA and RNA bases, and the interaction energy between two bonded complementary nucleobases is dependent on the intrinsic basicity and acidity of the acceptor and donor groups. In addition, understanding the intrinsic reactivity of nucleic bases can shed light on key biosynthetic mechanisms for which nucleobases are substrates. In this review, we highlight advances in our lab toward understanding the fundamental reactivity of DNA and RNA. In particular, we focus on our investigation of the gas phase acidities and basicities of natural and unnatural nucleobases, and the implications of our results for the mechanisms of nucleotide biosynthetic and repair enzymes.     

A simple model of DNA denaturation and mutually avoiding walks statistics

Recently Garel, Monthus and Orland [Europhys. Lett. 55, 132 (2001)] considered a model of DNA denaturation in which excluded volume effects within each strand are neglected, while mutual avoidance is included. Using an approximate scheme they found a first order denaturation. We show that a first order transition for this model follows from exact results for the statistics of two mutually avoiding random walks, whose reunion exponent is c > 2, both in two and three dimensions. Analytical estimates of c due to the interactions with other denaturated loops, as well as numerical calculations, indicate that the transition is even sharper than in models where excluded volume effects are fully incorporated. The probability distribution of distances between homologous base pairs decays as a power law at the transition. Received 8 July 2002 / Received in final form 25 July 2002 Published online 17 September 2002     

Silencing IL-23 expression by a small hairpin RNA protects against asthma in mice

To determine the impact of IL-23 knockdown by RNA interference on the development and severity of ovalbumin (OVA)-induced asthmatic inflammation, and the potential mechanisms in mice, the IL-23-specific RNAi-expressing pSRZsi-IL-23p19 plasmid was constructed and inhaled into OVA-sensitized mice before each challenge, as compared with that of control mice treated with alum or budesonide. Inhalation of the pSRZsi-IL-23p19, significantly reduced the levels of OVA-challenge induced IL-23 in the lung tissues by nearly 75%, determined by RT-PCR. In addition, knockdown of IL-23 expression dramatically reduced the numbers of eosinophils and neutrophils in BALF and mitigated inflammation in the lungs of asthmatic mice. Furthermore, knockdown of IL-23 expression significantly decreased the levels of serum IgE, IL-23, IL-17, and IL-4, but not IFNgamma, and its anti-inflammatory effects were similar to or better than that of treatment with budesonide in asthmatic mice. Our data support the notion that IL-23 and associated Th17 responses contribute to the pathogenic process of bronchial asthma. Knockdown of IL-23 by RNAi effectively inhibits asthmatic inflammation, which is associated with mitigating the production of IL-17 and IL-4 in asthmatic mice.     

Two-temperature hybridization for microarray detection of label-free microRNAs with attomole detection and superior specificity

Two is better than one: Two short locked nucleic acid based probes were used to collectively capture and detect microRNAs by a simple two-temperature hybridization process. Intact microRNAs were directly measured down to attomolar concentrations with a high specificity and nearly four orders of magnitude of dynamic range. Single base mismatches in the microRNAs were potently discriminated from the perfectly matched targets.     

Plug and play with RNA

Retooling RNA: RNA aptamers are high-affinity ligands that can be assembled with other structures to yield multivalent molecules. These properties have been addressed in two recent studies: One describes a GFP-like RNA reporter used to study the dynamics of endogenous RNA; the other study reports on an aptamer-templated assembly of multi-enzyme complexes in bacteria for the controlled production of secondary molecules (see picture).