Characterization of the promoter region of the murine Catsper2 gene.

CATSPER2 (Cation channel sperm-associated protein 2) protein, which is part of the calcium CATSPER channel located in the membrane of the flagellar principal piece of the sperm cell, is only expressed in the testis during spermatogenesis. Deletions or mutations in the Catsper2 gene are associated with the deafness-infertility syndrome (DIS) and non-syndromic male infertility. However, the mechanisms by which Catsper2 is regulated are unknown. Here, we report the characterization of the promoter region of murine Catsper2 and the role of CTCF and CREMτ in its transcription. We report that the promoter region has transcriptional activity in both directions, as determined by observing luciferase activity in mouse Sertoli and GC-1 spg transfected cells. WGBS data analysis indicated that a CpG island identified in silico is non-methylated; Chromatin immunoprecipitation (ChIP)-seq data analysis revealed that histone marks H3K4me3 and H3K36me3 are present in the promoter and body of the Catsper2 gene respectively, indicating that Catsper2 is subject to epigenetic regulation. In addition, the murine Catsper2 core promoter was delimited to a region between -54/+189 relative to the transcription start site (TSS), where three CTCF and one CRE binding site were predicted. The functionality of these sites was determined by mutation of the CTCF sites and deletion of the CRE site. Finally, ChIP assays confirmed that CREMτ and CTCF bind to the Catsper2 minimal promoter region. This study represents the first functional analysis of the murine Catsper2 promoter region and the mechanisms that regulate its expression.

Inhibition of Human Papillomavirus Type 16 Infection Using an RNA Aptamer.

Human papillomavirus type 16 (HPV16) DNA has been found in ∼50% of cervical tumors worldwide. HPV infection starts with the binding of the virus capsid to heparan sulfate (HS) receptors exposed on the surface of epithelial basal layer keratinocytes. Previously, our group isolated a high-affinity RNA aptamer (Sc5c3) specific for HPV16 L1 virus-like particles (VLPs). In this study, we report the inhibition of HPV16 infection by Sc5c3 in a pseudovirus (PsVs) model. 293TT cells were infected by HPV16 PsVs containing the yellow fluorescent protein (YFP) as reporter gene. Incubation of HPV16 PsVs with Sc5c3 before infection resulted in a dose-dependent decrease in YFP fluorescence, suggesting infection inhibition. Aptamer degradation by RNase A restored PsVs infectivity, supporting the previous observation that Sc5c3 aptamer can inhibit infection. VLP mutants with removed HS binding sites were used in binding assays to elucidate the Sc5c3 blocking mechanism; however, no binding difference was observed between wild-type and mutant VLPs, suggesting that pseudoinfection inhibition relies on mechanisms additional to electrostatic HS binding site interaction. A DNA/RNA Sc5c3 version also inhibited HPV PsVs infection, suggesting that a modified, nuclease-resistant Sc5c3 may be used to inhibit HPV16 infection in vivo.

Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention.

Targeting of the HPV-16 E7 protein by RNA aptamers.

The expression of high-risk human papillomavirus E6 and E7 proteins in most cervical tumors raised a considerable interest in the diagnostic and therapeutic applications of functional oligonucleotides (i.e., DNAzymes, ribozymes, and aptamers) directed against HPV targets. Aptamers are short single-stranded oligonucleotides that specifically recognize a wide variety of molecular targets, including HPV proteins. Here, we describe a protocol for the successful isolation of RNA aptamers directed at the recombinant HPV-16 E7 protein through the application of the SELEX method. Once the nucleic acid sequence of a functional aptamer is determined, large amounts of the oligonucleotide can be produced and modified at low cost and high efficiency. The remarkable affinity and specificity of aptamers for their targets make these molecules the next-generation tool for diagnostics and therapeutics of cervical cancer.

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Inhibition of human papillomavirus expression using DNAzymes.

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

A triplex ribozyme expression system based on a single hairpin ribozyme.

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

Advances in the development of ribozymes and antisense oligodeoxynucleotides as antiviral agents for human papillomaviruses.

Urogenital human papillomavirus (HPV) infections are the most common viral sexually transmitted disease in women. On a worldwide basis cervical cancer is the second most prevalent cancer of women. Although HPV infection is not sufficient to induce cancer, the causal relation between high-risk HPV infection and cervical cancer is well established. Over 99% of cervical cancers are positive for high-risk HPV. Therefore, there is a need for newer approaches to treat HPV infection. Two novel approaches for inactivating gene expression involve ribozymes and oligonucleotides. Methods for identification of target genes involved in neoplastic transformation and tumour growth have been established, and these will lead to therapeutic approaches without any damage to normal cellular RNA molecules, which is often associated with conventional therapeutics. Ribozymes and oligonucleotides represent rational antiviral approaches for inhibiting the growth of cervical lesions and carcinomas by interfering with E6/E7 RNA production. The E6 and E7 genes of high-risk HPVs cooperate to immortalize primary epithelial cells and because they are found in cervical cancer are considered the hallmark of cervical cancer. The use and modification of ribozymes and antisense oligodeoxynucleotides can inhibit the growth of HPV-16 and HPV-18 immortalized cells, and tumour cells by eliminating E6/E7 transcript. Hammerhead and hairpin ribozymes have been widely studied because of their potential use for gene therapy and their place as therapeutic tools for cervical cancer is being evaluated. Although antiviral ribozymes and antisense molecules have been effective as in vitro or in vivo inhibitors of high-risk HPV-positive cells, none is currently in clinical trial. There are, however, a number of other antisense therapies in Phase I-III clinical trial for several oncogenes.