title: DNA aptamers selectively target Leishmania infantum H2A protein.
abstract: Parasites of the genus Leishmania produce leishmaniasis which affects millions people around the world. Understanding the molecular characteristics of the parasite can increase the knowledge about the mechanisms underlying disease development and progression. Thus, the study of the molecular features of histones has been considered of particular interest because Leishmania does not condense the chromatin during mitosis and, consequently, a different role for these proteins in the biology of the parasite can be expected. Furthermore, the sequence divergences in the amino and in the carboxy-terminal domains of the kinetoplastid core histones convert them in potential diagnostic and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania histones is essential for the progress of this kind of study. Two aptamers which specifically recognize Leishmania infantum H2A histone were cloned from a previously obtained ssDNA enriched population. These aptamers were sequenced and subjected to an in silico analysis. ELONA, slot blot and Western blot were performed to establish aptamer affinity and specificity for LiH2A histone and ELONA assays using peptides corresponding to overlapped sequences of LiH2A were made mapping the aptamers:LiH2A interaction. As "proofs of concept", aptamers were used to determine the number of parasites in an ELONA platform and to purify LiH2A from complex mixtures. The aptamers showed different secondary structures among them; however, both of them were able to recognize the same peptides located in a side of the protein. In addition, we demonstrate that these aptamers are useful for LiH2A identification and also may be of potential application as diagnostic system and as a laboratory tool with purification purpose.
authors.affiliation: Departamento de Bioquímica-Investigación, Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Ramón y Cajal, Madrid, Spain.
authors.forename: M Elena
authors.initials: ME
authors.lastname: Martín
authors.forename: Marta
authors.initials: M
authors.lastname: García-Hernández
authors.forename: Eva M
authors.initials: EM
authors.lastname: García-Recio
authors.forename: Gerónimo F
authors.initials: GF
authors.lastname: Gómez-Chacón
authors.forename: Marta
authors.initials: M
authors.lastname: Sánchez-López
authors.forename: Víctor M
authors.initials: VM
authors.lastname: González
mesh_terms: D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D018314:Leishmania infantum / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D052157:Aptamers, Nucleotide; D006657:Histones; D015800:Protozoan Proteins