Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides

We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°Nm, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4′-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4′-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity

Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes

<div><p>Background</p><p>Insecticide resistance in the housefly, <i>Musca domestica</i>, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes.</p><p>Results</p><p>The <i>de novo</i> assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in <i>cyp6d1</i>. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools.</p><p>Conclusion</p><p>Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification.</p></div

Motifs found in the 1000 bp upstream of promoter of selected CYPs P450 in the MEME analysis.

<p>The combination of TOMTOM and GOMO provides information regarding the novelty of the motif and the probability that this motif is involved in transcription regulation. The found motifs were presented with their e-value. GOMO is used to provide information on what type of GO term could be associated to this DNA motif using the <i>Drosophila melanogaster</i> sequence as reference. The TOMTOM hits provide information on the number of known transcription factor to which this motif is sequence wise close using Pearson correlation coefficient with E-value <10. The <i>p</i> value is the probability that the match occurred by random chance according to the null model, E value is the expected number of false positives in the matches up to this point and q value is the minimum <b>F</b>alse <b>D</b>iscovery <b>R</b>ate required to include the match.</p

Motifs found in the mRNA of selected CYPs P450 in the MEME analysis.

<p>The combination of TOMTOM and GOMO provides information regarding the novelty of the motif and the probability that this motif is involved in transcription regulation. The found motifs were presented with their e-value. GOMO is used to provide information on what type of GO term could be associated to this DNA motif using the <i>Drosophila melanogaster</i> sequence as reference. The TOMTOM hits provide information on the number of known transcription factor to which this motif is sequence wise close Pearson correlation coefficient with E-value <10. The <i>p</i> value is the probability that the match occurred by random chance according to the null model, E value is the expected number of false positives in the matches up to this point and q value is the minimum <b>F</b>alse <b>D</b>iscovery <b>R</b>ate required to include the match.</p

Summary of run statistics and assembly.

<p>Summary of run statistics and assembly.</p