In vitro selection of RNA aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein 1

The var-gene encoding Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is known to play a major role in the pathogenicity of the P. falciparum parasite. The protein enables the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing clearance from the bloodstream. The development and spread of resistance towards most anti-malarial drugs used for treatment and prevention of the most severe form of malaria truly emphasise the importance of a continuous research and development of new drugs. In this study we use Systematic Evolution of Ligands by EXponential enrichment (SELEX) methodology to isolate high-affinity ligands (aptamers). To validate the results from the SELEX in vitro selection, different aptamers have been selected against PfEMP1 in a live cell assay of P. falciparum strain FCR3S1.2, a highly rosetting strain. We have been able to show the rosette disrupting capacity of these SELEX-aptamers at concentrations of 33 nM and with 100% disruption at 387 nM. The described results show that RNA aptamers are promising candidates for adjunct therapy in severe malaria

The 'PUCE CAFE' Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits

Background: Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results: The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion: We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Genoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research

Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

<p>Abstract</p> <p>Background</p> <p>Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications.</p> <p>Results</p> <p>Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a fluorescent sandwich assay on the surface of magnetic microbeads is also demonstrated.</p> <p>Conclusions</p> <p>This article catalogues numerous DNA aptamer sequences which can bind various important pathogenic arboviruses and have, in some cases, already demonstrated diagnostic potential. These aptamer sequences are proprietary, patent-pending, and partially characterized. Therefore, they are offered to the scientific community for potential research use in diagnostic assays, biosensor applications or for possible passive immunity and prophylaxis against pathogenic viruses.</p