Antisense tools for functional studies of human Argonaute proteins.

The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.

Oligomeric nucleic acids as antivirals.

Based on the natural functions and chemical characteristics of nucleic acids, a variety of novel synthetic drugs and tools to explore biological systems have become available in recent years. To date, a great number of antisense oligonucleotides, RNA interference-based tools, CpG­containing oligonucleotides, catalytic oligonucleotides, decoys and aptamers has been produced synthetically and applied successfully for understanding and manipulating biological processes and in clinical trials to treat a variety of diseases. Their versatility and potency make them equally suited candidates for fighting viral infections. Here, we describe the different types of nucleic acid-based antivirals, their mechanism of action, their advantages and limitations, and their future prospects.

Specific binding of a hexanucleotide to HIV-1 reverse transcriptase: a novel class of bioactive molecules.

Short oligonucleotides below 8-10 nt in length adopt relatively simple structures. Accordingly, they represent interesting and so far unexplored lead compounds as molecular tools and, potentially, for drug development as a rational improvement of efficacy seem to be less complex than for other classes of longer oligomeric nucleic acid. As a 'proof of concept', we describe the highly specific binding of the hexanucleotide UCGUGU (Hex-S3) to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) as a model target. Ultraviolet (UV) cross-linking studies and competition experiments with primer/template substrates and a RT-directed aptamer suggest site-specific binding of Hex-S3 to the large subunit (p66) of the viral enzyme. The affinity of 5.3 muM is related to hexanucleotide-specific suppression of HIV-1 replication in human cells by up to three orders of magnitude indicating that Hex-S3 exerts specific and biologically relevant activity. Experimental evidence described here further suggests a systematic hexamer array-based search for new tools for molecular biology and novel lead compounds in nucleic acid-based drug development.

A human folliculoid microsphere assay for exploring epithelial- mesenchymal interactions in the human hair follicle.

The search for more effective drugs for the management of common hair growth disorders remains a top priority, both for clinical dermatology and industry. In this pilot study, we report a pragmatic organotypic assay for basic and applied hair research. The patented technique produces microdroplets, which generate human folliculoid microspheres (HFMs), consisting of human dermal papilla fibroblasts and outer root sheath keratinocytes within an extracellular matrix that simulates elements of the hair follicle mesenchyme. Studying a number of different markers (for example, proliferation, apoptosis, cytokeratin-6, versican), we show that these HFMs, cultured under well-defined conditions, retain several essential epithelial-mesenchymal interactions characteristic for human scalp hair follicle. Selected, recognized hair growth-modulatory agents modulate these parameters in a manner that suggests that HFMs allow the standardized preclinical assessment of test agents on relevant human hair growth markers under substantially simplified in vitro conditions that approximate the in vivo situation. Furthermore, we show by immunohistochemistry, reverse transcriptase-PCR, and DNA microarray techniques that HFMs also offer a useful discovery tool for the identification of target genes and their products for candidate hair drugs. HFM thus represent an instructive modern experimental and screening tool for basic and applied hair research in the human system.

Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery.

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.

A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.

The vanilloid receptor-1 (VR1, or transient receptor potential vanilloid-1 receptor, TRPV1) is activated by capsaicin, the key ingredient of hot peppers. TRPV1 was originally described on sensory neurons as a central integrator of various nociceptive stimuli. However, several human skin cell populations are also now recognized to express TRPV1, but with unknown function. Exploiting the human hair follicle (HF) as a prototypic epithelial-mesenchymal interaction system, we have characterized the HF expression of TRPV1 in situ and have examined TRPV1 signaling in organ-cultured human scalp HF and outer root sheath (ORS) keratinocytes in vitro. TRPV1 immunoreactivity was confined to distinct epithelial compartments of the human HF, mainly to the ORS and hair matrix. In organ culture, TRPV1 activation by capsaicin resulted in a dose-dependent and TRPV1-specific inhibition of hair shaft elongation, suppression of proliferation, induction of apoptosis, premature HF regression (catagen), and up-regulation of intrafollicular transforming growth factor-beta(2). Cultured human ORS keratinocytes also expressed functional TRPV1, whose stimulation inhibited proliferation, induced apoptosis, elevated intracellular calcium concentration, up-regulated known endogenous hair growth inhibitors (interleukin-1beta, transforming growth factor-beta(2)), and down-regulated known hair growth promoters (hepatocyte growth factor, insulin-like growth factor-I, stem cell factor). These findings strongly support TRPV1 as a significant novel player in human hair growth control, underscore the physiological importance of TRPV1 in human skin beyond nociception, and identify TRPV1 as a promising, novel target for pharmacological manipulations of epithelial growth disorders.