On the complexity of resource-bounded logics

We revisit decidability results for resource-bounded logics and use decision problems for vector addition systems with states (VASS) to characterise the complexity of (decidable) model-checking problems. We show that the model-checking problem for the logic RB+-ATL is 2EXPTIME-complete by using recent results on alternating VASS. In addition, we establish that the model-checking problem for RBTL is decidable and has the same complexity as for RBTL* (the extension of RBTL with arbitrary path formulae), namely EXPSPACE-complete, proving a new decidability result as a by-product of the approach. Finally, we establish that the model-checking problem for RB+-ATL* is decidable by a reduction to parity games, and show how to synthesise values for resource parameters

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0°C) than the wild-type double-stranded DNA (dsDNA, 26.0°C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0°C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2′-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0°C) than duplexes with the complementary ssDNAs (31.5 and 19.5°C). The excimer band observed in the ds2′-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2′-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2′-OMe-RNA

Enzymatic incorporation of LNA nucleotides

The present invention relates to oligonucleotides comprising LNA units and enzymatic methods of synthesizing such oligonucleotides. Oligonucleotide comprising LNA units have various characteristics, e.g. improved biostability and increased > affinity for complementary oligonucleotides. The methods may be part of detection reactions, polymerase chain reaction, RNA transcription or in vitro evolution processes such as SELEX

Enzymatic incorporation of LNA nucleotides

The present invention relates to oligonucleotides comprising LNA units and enzymatic methods of synthesizing such oligonucleotides. Oligonucleotide comprising LNA units have various characteristics, e.g. improved biostability and increased > affinity for complementary oligonucleotides. The methods may be part of detection reactions, polymerase chain reaction, RNA transcription or in vitro evolution processes such as SELEX

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

10.1371/journal.pone.0050964PLoS ONE81

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (k(obs )increased from 0.014 min(-1 )to 0.78 min(-1)). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers

Prebiotic potential of a maize-based soluble fibre and impact of dose on the human gut microbiota

Dietary management of the human gut microbiota towards a more beneficial composition is one approach that may improve host health. To date, a large number of human intervention studies have demonstrated that dietary consumption of certain food products can result in significant changes in the composition of the gut microbiota i.e. the prebiotic concept. Thus the prebiotic effect is now established as a dietary approach to increase beneficial gut bacteria and it has been associated with modulation of health biomarkers and modulation of the immune system. Promitor™ Soluble Corn Fibre (SCF) is a well-known maize-derived source of dietary fibre with potential selective fermentation properties. Our aim was to determine the optimum prebiotic dose of tolerance, desired changes to microbiota and fermentation of SCF in healthy adult subjects. A double-blind, randomised, parallel study was completed where volunteers (n = 8/treatment group) consumed 8, 14 or 21 g from SCF (6, 12 and 18 g/fibre delivered respectively) over 14-d. Over the range of doses studied, SCF was well tolerated Numbers of bifidobacteria were significantly higher for the 6 g/fibre/day compared to 12g and 18g/fibre delivered/day (mean 9.25 and 9.73 Log10 cells/g fresh faeces in the pre-treatment and treatment periods respectively). Such a numerical change of 0.5 Log10 bifidobacteria/g fresh faeces is consistent with those changes observed for inulin-type fructans, which are recognised prebiotics. A possible prebiotic effect of SCF was therefore demonstrated by its stimulation of bifidobacteria numbers in the overall gut microbiota during a short-term intervention

Ultrafast Structural Dynamics of Photo-Reactions Revealed by Model-Independent X-ray Cross-Correlation Analysis

We applied angular X-ray Cross-Correlation analysis (XCCA) to scattering images from a femtosecond resolution LCLS X-ray free-electron laser (XFEL) pump-probe experiment with solvated PtPOP ([Pt$_2$(P$_2$O$_5$H$_2$)$_4$]$^{4-}$) metal complex molecules. The molecules were pumped with linear polarized laser pulses creating an excited state population with a preferred orientational (alignment) direction. Two time scales of $1.9\pm1.5$ ps and $46\pm10$ ps were revealed by model-independent XCCA, associated with an internal structural changes and rotational dephasing, respectively. Our studies illustrate the potential of XCCA to reveal hidden structural information in a model independent analysis of time evolution of solvated metal complex molecules.Comment: 8 pages, 5 figures, 50 reference

Site-specific O-glycosylation on the MUC_{2} mucin protein inhibits cleavage by the porphyromonas gingivalis secreted cysteine protease (RgpB)

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation