In Vitro Selection of Leishmania infantum H3-Binding ssDNA Aptamers Edurne Ramos, 1 Miguel Moreno, 2 M. Elena Martı ´n, 1 Manuel Soto, 3 and Vı ´ctor M. Gonzalez 1 Aptamers are single-stranded DNA or RNA oligonucleotides that adopt specific three-dimensional structures binding with high affinity and specificity to their targets. These molecules are being currently used with de- tection and diagnosis purposes. Parasites of the genus Leishmania cause leishmaniosis in humans and animals. Interestingly, Leishmania do not condense their chromatin during mitosis, and histone genes could be responsible for this fact. Although histones are extremely conserved proteins, reflecting their apparent universality of function, sequence similarity of kinetoplastid core histones with that of higher eukaryotes is found predomi- nantly in the globular region. However, high sequence divergences in the N-terminal and C-terminal domains are found that convert them into potential diagnostic and/or therapeutics targets. We have successfully isolated a pool of DNA aptamers, named SELH3, which binds to Leishmania infantum H3 with high affinity and speci- ficity. Thus, it appears that this novel anti-H3 aptamer population may be of potential application as a diagnostic system for leishmaniosis. Introduction A ptamers are structured single-stranded nucleic acids, selected from combinatorial libraries by systematic evolution of ligands by exponential enrichment (SELEX) technology, that selectively bind target molecules with high affinity and specificity (Ellington and Szostak, 1990; 1992; Tuerk and Gold, 1990). Aptamers can be raised against a wide range of different targets including small molecules to highly complex proteins or whole cells and viruses (Bock et al., 1992; Green et al., 1995; Famulok, 1999; Moreno et al., 2003; Vive- kananda and Kiel, 2006). Due to the highly defined tertiary structures of the aptamers, they are able to form stable and specific complexes with the targets in a similar way that an- tibodies do. At this respect, comparisons of aptamers with antibodies that bind to the same targets have shown that both of them use similar strategies for the formation of well- defined binding patterns ( Marshall et al., 1997; Hermann and Patel, 2000). In addition, aptamers are analogs to antibodies in specificity and affinity and very frequently form complexes that have dissociation constants in the nanomolar range and can clearly distinguish between even closely related protein targets ( Jenison et al., 1994; Uphoff et al., 1996). Aptamers offer, moreover, other advantages over antibody-based af- finity due to the nature of nucleic acids, which provides in- creased stability, easy regeneration, and simple modifications with fluorescent or other reporters during their synthesis for detection and neutralization. The selection process implies the incubation of a target with a library of nucleic acids, typically ranging from 10 14 to 10 18 different sequences. The target–nucleic acid complexes are isolated, and the nucleic acid molecule is amplified for a next round of selection. The process is repeated until the desired sample is enriched with sequences that display high affinity and specificity for the target of interest. In this way, it is often possible to identify aptamers with very high affinities (na- nomolar or subnanomolar) that can be considered potential biorecognition molecules. The causative agent of Leishmaniasis is a parasitic protozoa of the genus Leishmania that is transmitted to humans by sandflies. Most forms of leishmaniasis are originally infections of small mammals (‘‘reservoir hosts’’), which play a major role in the epidemiology of the disease. Sandflies become infected by ingesting blood from infected reservoir hosts or from infected people. Leishmania parasites possess a digenetic life cycle with 2 discrete morphological phases: the promastigote, which develops extracelullarly within the gut of the insect vector and the amastigote, which is specialized to sur- vive within the macrophage’s phagolysosome of the verte- brate host. These parasites, like other related kinetoplastid 1 Departamento de Bioquı ´mica-Investigacio ´ n, Instituto Ramo ´ n y Cajal de Investigacio ´ n Sanitaria (IRyCIS), Madrid, Spain. 2 Centro de Astrobiologı ´a (CSIC-INTA), Madrid, Spain. 3 Centro de Biologı ´a Molecular Severo Ochoa, Departamento de Biologı ´a Molecular, Universidad Auto ´ noma de Madrid, CSIC-UAM, Madrid, Spain. OLIGONUCLEOTIDES Volume 20, Number 4, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089/oli.2010.0240 207