USEFULNESS OF A RECURSIVE PCR TO INSERT A HIGH AFFINITY APTAMER INTO THE CALMODULIN MINOR SPACER of Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness in Panama. In this parasite, regulation of gene expression is mainly a post transcriptional process. Has been suggested that RNA-binding proteins have a key role in this gene regulation. Calmodulin is a highly conserved calcium sensor protein, encodes by three genes separated by two intergenic spacers (major and minor spacer). The study of molecular basis that led RNA-proteins interactions in T. cruzi could contribute to understand the biology of the parasite to develop novel strategies for disease control. We developed a methodology based on a recursive PCR to insert a streptavidin high affinity aptamer into the sequence of the minor spacer, as the basis for the development of a protein capture system. The complete calmodulin locus sequence was download from GenBank (AAHK01001263.1). Previous reported aptameric sequence (83bp) was manipulated for primer design. Sequences were edited with UGENE bioinformatic software. Calmodulin minor spacer (CMS) sequence was fragmented into two regions. Primers were designed to flank the two regions, the inner forward/reverse oligos attached to half aptamer sequences each. Final recursive PCR was able to amplify the Calmodulin minor spacer incorporating the aptamer. Sequence was confirmed by Sanger sequencing. Recursive PCR represent a useful tool to insert high affinity sequences as aptamers to study specific protein-RNA interactions.