PD-loop: A complex of duplex DNA with an oligonucleotide

A stable complex between duplex DNA and an oligonucleotide is assembled with the aid of a DNA synthetic mimic, peptide nucleic acid (PNA). Homopyrimidine PNAs are known to invade into short homopurine tracts in duplex DNA forming P-loops. We have found that P-loops, formed at two closely located purine tracts in the same DNA strand separated by a mixed purine–pyrimidine sequence, merge and open the double helix between them. The opposite DNA strand, which is not bound with PNA, exposes and becomes accessible for complexing with an oligonucleotide via Watson–Crick pairing. As a result, the PD-loop emerges, which consists of locally open duplex DNA, PNA “openers,” and an oligonucleotide. The PD-loop stability and sequence specificity are demonstrated by affinity capture of duplex DNAs by using biotinylated oligonucleotides and streptavidin-covered magnetic beads. The type of complex formed by PNAs, an oligonucleotide and duplex DNA we describe, opens ways for development of various in vitro and in situ hybridization techniques with duplex DNA and may find applications in DNA nanotechnology and genomics

Anti-microRNA-21 Therapy on Top of ACE Inhibition Delays Renal Failure in Alport Syndrome Mouse Models

Col4a3−/− Alport mice serve as an animal model for renal fibrosis. MicroRNA-21 (miR-21) expression has been shown to be increased in the kidneys of Alport syndrome patients. Here, we investigated the nephroprotective effects of Lademirsen anti-miR-21 therapy. We used a fast-progressing Col4a3−/− mouse model with a 129/SvJ background and an intermediate-progressing F1 hybrid mouse model with a mixed genetic background, with angiotensin-converting enzyme inhibitor (ACEi) monotherapy in combination with anti-miR-21 therapy. In the fast-progressing model, the anti miR-21 and ACEi therapies showed an additive effect in the reduction in fibrosis, the decline of proteinuria, the preservation of kidney function and increased survival. In the intermediate-progressing F1 model, the anti-miR-21 and ACEi therapies individually improved kidney pathology. Both also improved kidney function and survival; however, the combination showed a significant additive effect, particularly for survival. RNA sequencing (RNA-seq) gene expression profiling revealed that the anti-miR-21 and ACEi therapies modulate several common pathways. However, anti-miR-21 was particularly effective at normalizing the expression profiles of the genes involved in renal tubulointerstitial injury pathways. In conclusion, significant additive effects were detected for the combination of anti-miR-21 and ACEi therapies on kidney function, pathology and survival in Alport mouse models, as well as a strong differential effect of anti-miR-21 on the renal expression of fibrotic factors. These results support the addition of anti-miR-21 to the current standard of care (ACEi) in ongoing clinical trials in patients with Alport syndrome

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases