The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5-4 degrees C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the T(m) by only <1 degree C per modification and the T(m) of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2'-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t(1/2) = approximately 1.5 h to t(1/2) = approximately 15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2'-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.

Activation of the hypoxia-inducible factor alpha-subunits, HIF-1alpha and HIF-2alpha, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF-1alpha vs. HIF-2alpha to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF-1alpha siRNA, showing no effect by HIF-2alpha knock-down. The same was found for the activation of reporter genes driven by hypoxia-responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF-2alpha knock-down, as observed in Hep3B and Kelly cells. In contrast to the EPO-HRE reporter, the complete EPO enhancer displayed dependency on HIF-2alpha regulation, indicating that additional cis-acting elements confer HIF-2alpha specificity within this region. In 786-0 cells lacking HIF-1alpha protein, the identified HIF-1alpha target genes were regulated by HIF-2alpha. Overexpression of the HIFalpha subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFalpha subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.

A 58-mer L-RNA ligand that binds to naturally occurring D-adenosine with a dissociation constant of 1.7 microM in solution was identified from a combinatorial library employing mirror-design. The corresponding D-RNA ligand shows identical binding affinity to L-adenosine. Reciprocal chiral specificity was also evident from ligand discrimination; the binding affinity of the L-RNA ligand for D-adenosine was 9000-fold greater than its affinity for L-adenosine and vice versa. While the D-RNA ligand was rapidly degraded in human serum, the L-RNA ligand displayed an extraordinary stability. This indicates the potential application of specifically designed L-RNA ligands as stable monoclonal antibody analogues and the development of highly stable L-ribozymes.

BackgroundSELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.MethodologyWe have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel.ConclusionsHigh affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease.