Sampling, Logistics, and Analytics of Urine for RT-qPCR-based Diagnostics.

Body fluids in the context of cancer diagnosis are the primary source of liquid biopsy, i.e., biomarker detection that includes blood and serum, urine, and saliva.

Transcriptome analyses of urine RNA reveal tumor markers for human bladder cancer: validated amplicons for RT-qPCR-based detection.

Non-invasive clinical diagnostics of bladder cancer is feasible via a set of chemically distinct molecules including macromolecular tumor markers such as polypeptides and nucleic acids. In terms of tumor-related aberrant gene expression, RNA transcripts are the primary indicator of tumor-specific gene expression as for polypeptides and their metabolic products occur subsequently. Thus, in case of bladder cancer, urine RNA represents an early potentially useful diagnostic marker.

Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro.

The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC.

RNAi revised--target mRNA-dependent enhancement of gene silencing.

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level.

Activity and stability of hammerhead ribozymes containing 2'-C-methyluridine: a new RNA mimic.

We propose 2'-C-methylnucleotides as a new class of 2'-modified RNA mimics. These analogues are expected to provide 2'-OH groups capable of reproducing the interactions observed in natural RNA and, due to the presence of the Me group, to possess increased stability towards nucleases. In this work, we investigate the catalytic activity and nuclease resistance of hammerhead ribozymes carrying 2'-C-methyluridines in positions 4 and 7 of the catalytic core.

Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression.

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest.

Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts.

The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts.

Technical improvements in the computational target search for antisense oligonucleotides.

A number of theoretical and experimental approaches to design biologically active antisense oligonucleotides (AS-ON) have proven their usefulness. This includes systematic computational strategies that are based on the understanding of antisense mechanisms. Here, we investigate in detail the relationship between computational parameters of the local target search for the theoretical design of AS-ON and the hit rate, that is, the biologic efficacy of AS-ON in cell culture.

Local RNA target structure influences siRNA efficacy: a systematic global analysis.

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics.

Evaluation of bacterial RNase P RNA as a drug target.

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit.

The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides.

The biological activity of siRNA seems to be influenced by local characteristics of the target RNA, including local RNA folding. Here, we investigated quantitatively the relationship between local target accessibility and the extent of inhibition of the target gene by siRNA. Target accessibility was assessed by a computational approach that had been shown earlier to be consistent with experimental probing of target RNA.

The role of target accessibility for antisense inhibition.

It appears that the application of antisense nucleic acids to drug development and gene function analysis are now established fields, and success in the use of antisense molecules has increased over recent years through modulating chemical and biological properties of oligonucleotides via the specific chemistry employed in their construction. In contrast, the targets of antisense nucleic acids are unchangeable RNA molecules, each with a defined strcuture. The local properties of the target represent a major limitation of the effectiveness of complementary nucleic acid inhibitors, and as such the search for appropriate local target sites for the invasion of an antisense strand is the focus of current research.