Aptamers as a promising approach for the control of parasitic diseases

Aptamers are short single-stranded RNA or DNA oligonucleotides that are capable of binding various biological targets with high affinity and specificity. Their identification initially relies on a molecular process named SELEX (Systematic Evolution of Ligands by EXponential enrichment) that has been later modified in order to improve aptamer sensitivity, minimize duration and cost of the assay, as well as increase target types. Several biochemical modifications can help to enhance aptamer stability without affecting significantly target interaction. As a result, aptamers have generated a large interest as promising tools to compete with monoclonal antibodies for detection and inhibition of specific markers of human diseases. One aptamer-based drug is currently authorized and several others are being clinically evaluated. Despite advances in the knowledge of parasite biology and host–parasite interactions from “omics” data, protozoan parasites still affect millions of people around the world and there is an urgent need for drug target discovery and novel therapeutic concepts. In this context, aptamers represent promising tools for pathogen identification and control. Recent studies have reported the identification of “aptasensors” for parasite diagnosis, and “intramers” targeting intracellular proteins. Here we discuss various strategies that have been employed for intracellular expression of aptamers and expansion of their possible application, and propose that they may be suitable for the clinical use of aptamers in parasitic infections. Keywords: Aptamer, Parasite control, Protozoan parasite, SELEX strateg

Advances on Aptamers against Protozoan Parasites

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

The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in <i>Entamoeba histolytica</i>

<div><p>In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of <i>Entamoeba histolytica</i>, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety <u>X</u>) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the <i>EhPgp5</i> mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in <i>E. histolytica</i>. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite.</p> </div

Molecular characteristics of predicted EhCFIm25.

<p>A) Comparative molecular organization of CFIm25 proteins from <i>E. histolytica</i> and human. Upper panel, Schematic representation. The scale at the top indicates the size in aa. Numbers at the right are relative to the initial methionine in each protein. Lower panel, ClustalW sequence alignment of Nudix box. Black box, identical aa; grey box, similar aa. X, any residue; U, hydrophobic residue. D) Three dimensional organization of CFIm25 proteins from <i>E. histolytica</i> (left) and human (right). 3D modeling of EhCFIm25 was obtained using crystal data from human CFIm25 as template. The Nudix box is in black color. The UniProt KnowledgeBase database accession number for each predicted protein is indicated.</p

Expression of the recombinant EhCFIm25.

<p>A) Expression of the 6x-His-labeled EhCFIm25 protein. Bacteria <i>E. coli</i> were transformed with pRSET<i>-EhCFIm25</i> plasmid and protein expression was induced by the addition of 1 mM IPTG for 3 h. Proteins extracts (30 µg) were separated through 10% SDS-PAGE and gels were stained with Coomassie blue. Lane 1, molecular weight; lane 2, non-induced bacterial extract; lane 3, IPTG-induced bacterial extract. B) Immunodetection of rEhCFIm25 polypeptide by Western blot assays using anti-6x His tag antibodies. Lane 1, non-induced bacterial extract (30 μg); lane 2, IPTG-induced bacterial extract (30 μg). C) Purification of the 6x-His-labeled EhCFIm25 protein through affinity chromatography using a Ni-NTA column. Lane 1, molecular weight; lane 2, IPTG-induced bacterial extract; lane 3, unbound proteins; lane 4, wash using 150 mM imidazole; lanes 5-8, elution with 250 mM imidazole. D) Immunodetection of rEhCFIm25 polypeptide by Western blot assays using specific rabbit antibodies anti-EhCFm25Im. Lane 1, non-induced bacterial extract; lane 2, IPTG-induced bacterial extract; lane 3, IPTG-induced bacterial extract and anti-6x His tag antibodies used as control. E) Immunodetection of purified rEhCFIm25 polypeptide by Western blot assays. Lane 1, anti-6x His tag antibodies; lane 2, specific rabbit antibodies anti-EhCFIm25; lane 3, pre-immune serum; lane 4, control without primary antibody. Arrowhead, EhCFIm25.</p

Expression of the putative EhCFIm25 in <i>E. histolytica</i> trophozoites.

<p>A and B) Western blot assays. A) Cytoplasmic (lanes 1, 3, 5 and 7) and nuclear (lanes 2, 4, 6 and 8) extracts of <i>E. histolytica</i> trophozoites were separated through 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane and incubated with antibodies. Lanes 1 and 2, anti-EhCFIm25 antibodies; lanes 3 and 4, pre-immune serum; lanes 5 and 6, anti-EhPAP antibodies; lanes 7 and 8, anti-EhPC4 antibodies. B) Cytoplasmic (lanes 1, 3, 5 and 7) and nuclear (lanes 2, 4, 6 and 8) extracts were treated with 5% β-mercaptoethanol (lanes 1 to 4) or 8% β-mercaptoethanol (lanes 5 to 8) and separated through 10% SDS-PAGE in the presence of 8 M urea, and electrotransferred to a nitrocellulose membrane before being incubated with antibodies. Lanes 1, 2, 5 and 6: anti-EhCFIm25 antibodies; lanes 3, 4, 7 and 8: pre-immune serum. Arrowhead, endogenous EhCFIm25. C) Primers to evidence mRNA expression of genes at locus EHI_077110 (up) and EHI_077000 bottom) in RT-PCR assays. D) RT-PCR assays. <i>EhCFIm25</i> transcript was PCR amplified using cDNA synthesized from total RNA and products were analyzed through ethidium bromide stained polyacrylamide gels. Lane 1, molecular size markers; lane 2, cDNA; lane 3, control using genomic DNA from <i>E. histolytica</i>; lane 4, control using oligonucleotides for <i>actin</i> gene; lane 5, control using pRSET<i>-EhCFIm25</i>; lane 6, control without cDNA.</p

RNA binding activity of rEhCFIm25.

<p>A) REMSA. rEhCFIm25 was incubated with [α-³²P] UTP-labeled PSIII¹⁵⁶ RNA probe (5×10⁵ cpm) at 4 °C for 15 min. Complexes were resolved through 6% non-denaturing PAGE and detected in a Phosphor Imager apparatus. Lane 1, free probe; lane 2, RNA probe plus 5 nM rEhCFIm25; lane 3, as in lane 2 plus specific competitor (Sc) (350-fold molar excess of unlabeled probe); lane 4, as in lane 2 plus unspecific competitor (Uc) (350-fold molar excess of unlabeled tRNA); lane 5, RNA probe plus 10 nM rEhCFIm25; lane 6, as in lane 2 plus proteinase K; lane 7, as in lane 2 plus RNAse; lanes 8 and 9, as in lane 2 plus antibody anti-EhCFIm25. Arrowhead, RNA-protein complex. B) Densitometry analysis of complexes detected in A. Pixels corresponding to C_I in lane 2 was taken as 100% and used to normalize data.</p

<i>In vitro</i> protein-protein interaction assays.

<p>A) Pull-Down assay. The rEhCFIm25 immobilized on Ni²⁺-NTA column was incubated with NE. After washing, proteins were eluted, separated though 10% SDS-PAGE and stained with Coomassie Brilliant blue. Lane 1, Molecular weight markers; lane 2, proteins not retained on the column; lanes 3 and 4, washing fraction; lane 5, elution fraction. B) Far-Western assay. Purified rEhCFIm25 (lanes 1 to 4) and rEhPAP (lanes 5 to 8) were subjected to 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane that was incubated with rEhPAP (lane 1) or rEhCFIm25 (lane 5). Proteins were immunodetected by specific antibody anti-EhPAP (lanes 1, 2 and 7) or anti-EhCFIm25 (lanes 3, 5 and 6) and revealed by the ECL Plus Western blotting system (Amersham). Anti-His antibody was used as control (lanes 4 and 8).</p