Bulge-Forming miRNases Cleave Oncogenic miRNAs at the Central Loop Region in a Sequence-Specific Manner.

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)(2)Gly](2) (BC–miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC–miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA. A thorough study of mono- and bis-BC–miRNases (containing one or two catalytic peptides, respectively) revealed that: (i) the sequence of miRNA bulge loops and neighbouring motifs are of fundamental importance for efficient miRNA cleavage (i.e., motifs containing repeating pyrimidine–A bonds are more susceptible to cleavage); (ii) the incorporation of the second catalytic peptide in the same molecular scaffold increases the potency of BC–miRNase, providing a complete degradation of miR-17 within 72 h; (iii) the synergetic co-operation of BC–miRNases with RNase H accelerates the rate of miRNA catalytic cleavage by both the conjugate and the enzyme. Such synergy allows the rapid destruction of constantly emerging miRNA to maintain sufficient knockdown and achieve a desired therapeutic effect

Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment.

Taking into account recently obtained data indicating the participation of circulating extracellular DNA (exDNA) in tumorigenesis, enzymes with deoxyribonucleic activity have again been considered as potential antitumour and antimetastatic drugs. Previously, using murine Lewis lung carcinoma and hepatocellular carcinoma A1 tumour models, we have shown the antimetastatic activity of bovine DNase I, which correlates with an increase of DNase activity and a decrease of exDNA concentration in the blood serum of tumour-bearing mice. In this work, using next-generation sequencing on the ABS SOLiD™ 5.500 platform, we performed a search for molecular targets of DNase I by comparing the exDNA profiles of healthy animals, untreated animals with Lewis lung carcinoma (LLC) and those with LLC treated with DNase I. We found that upon DNase I treatment of LLC-bearing mice, together with inhibition of metastasis, a number of strong alterations in the patterns of exDNA were observed. The major differences in exDNA profiles between groups were: i) the level of GC-poor sequences increased during tumour development was reduced to that of healthy mice; ii) levels of sequences corresponding to tumour-associated genes Hmga2, Myc and Jun were reduced in the DNase I-treated group in comparison with non-treated mice; iii) 224 types of tandem repeat over-presented in untreated LLC-bearing mice were significantly reduced after DNase I treatment. The most important result obtained in the work is that DNase I decreased the level of B-subfamily repeats having homology to human ALU repeats, known as markers of carcinogenesis, to the level of healthy animals. Thus, the obtained data lead us to suppose that circulating exDNA plays a role in tumour dissemination, and alteration of multiple molecular targets in the bloodstream by DNase I reduces the invasive potential of tumours

Abundance of fragments encoding tumour-associated genes <i>Hmga2</i>, <i>Fos</i>, <i>Myc</i>, <i>Nras</i> and <i>Jun</i> in DNA libraries.

<p>Abundance of fragments encoding tumour-associated genes <i>Hmga2</i>, <i>Fos</i>, <i>Myc</i>, <i>Nras</i> and <i>Jun</i> in DNA libraries.</p

Abundance of tandem repeats belonging to 11 major subfamilies in DNA libraries.

<p>Abundance of tandem repeats belonging to 11 major subfamilies in DNA libraries.</p

Concentration of exDNA and DNA libraries at different stages of construction.

<p>Concentration of exDNA and DNA libraries at different stages of construction.</p

Heatmap on the abundance of B-family tandem repeats homologous to human ALU repeats in DNA libraries L_h1, L_h2, L_LLC and L_D.

<p>The decreased and increased tandem repeats are indicated by range of red and green intensities, respectively. Dendrograms were derived by complete linkage clustering of tandem repeats or samples using Euclidean distances between scaled log-transformed RPKM values (Colour Key), respectively.</p