Advances on Aptamers against Protozoan Parasites

Abstract

Aptamers are single-stranded DNA or RNA sequences with a unique three-dimensional structure that allows them to recognize a particular target with high affinity. Although their specific recognition activity could make them similar to monoclonal antibodies, their ability to bind to a large range of non-immunogenic targets greatly expands their potential as tools for diagnosis, therapeutic agents, detection of food risks, biosensors, detection of toxins, drug carriers, and nanoparticle markers, among others. One aptamer named Pegaptanib is currently used for treating macular degeneration associated with age, and many other aptamers are in different clinical stages of development of evaluation for various human diseases. In the area of parasitology, research on aptamers has been growing rapidly in the past few years. Here we describe the development of aptamers raised against the main protozoan parasites that affect hundreds of millions of people in underdeveloped and developing countries, remaining a major health concern worldwide, i.e. Trypanosoma spp., Plasmodium spp., Leishmania spp., Entamoeba histolytica, and Cryptosporidium parvuum. The latest progress made in this area confirmed that DNA and RNA aptamers represent attractive alternative molecules in the search for new tools to detect and treat these parasitic infections that affect human health worldwide.

New directions in antimalarial target validation

“…After binding to their target sequence on each side of the chromosome, the zincfinger proteins induce nuclease activity, causing a double-strand break (DSB). This allows for alteration of the target DNA sequence by taking advantage of the homology-directed DNA repair mechanisms at the site of nuclease action [65] (Figure 1(c)). In both P. falciparum and P. vivax, experiments have demonstrated the generation of ZFN-mediated gene deletions, allelic exchanges, and specific nucleotide alterations in the presence or absence of selectable markers (Table 1).…”

“…Conditional knockdown at protein level can be obtained by the introduction of destabilizing domains to the protein of interest. Integration of FK506-binding protein (FKBP)-based destabilization domain (DD) [88] or an Escherichia coli DHFR destabilizing domain (DDD) [65,89] in fusion with the target protein leads to protein ubiquitylation and degradation by Plasmodium degradation machinery due to instability of these domains. However, this instability can be reversed by the addition of stabilizing compounds (Shield 1 for DD fusion proteins and trimethoprim for DDD fusion proteins), which allows for control of the expression at protein level.…”

“…The conditional expression of these enzymes, by systems like the tet-OFF described above, allow for conditional knockout of Plasmodium essential genes. Initial studies using Cre recombinase, which recognizes loxP sites, have shown low efficiency when combined with a Tet promoter in P. falciparum, while FLP, which recognizes frt sites, provided better results [65,103]. However, the more recent development of a split diCre protein, which can be induced to dimerize into a functional enzyme by the addition of the ligand rapamycin, has provided control of the expression levels in a more precise manner [104].…”

“…Aptamers are single-stranded RNA (ssRNA) or single-stranded DNA (ssDNA) oligonucleotides whose unique threedimensional structure enables them to interact with a specific target [65]. The use of aptamers, also known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX), consists of incubation of a random nucleic acid library with the target culture/lysate followed by isolation of target-bound aptamers and amplification of these aptamers using conserved primer sequences for further rounds of selection [107,108] (Figure 3 (a)).…”

“…Schematic representation of conditional/inducible validation techniques applied to Plasmodium. (a) Post-translational knockdown principle in Plasmodium.Integration of FK506-binding protein (FKBP)-based destabilization domain (DD) or an Escherichia coli DHFR destabilizing domain (DDD) in fusion with the target protein leads to protein degradation by Plasmodium degradation machinery when stabilizing compounds are removed from the culture[65,89]. (b) Tet-OFF system.…”