e8afa5ced1be978a1e2ff84f26b7ce58 pmc_9695703.pdf 02f47d52b69604620d4d92fdedd0033e994596d9 pmc_9695703.pdf 5657c860db987eba0e3cdcff63ecabb588c2e1046b396975e51d13f94bba1fa9 pmc_9695703.pdf Title: Selection of an Aptamer against the Enzyme 1-deoxy-D-xylulose-5-phosphate Reductoisomerase from Plasmodium falciparum Subject: The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 M a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naïve erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches. Keywords: Plasmodium; DNA aptamers; methyl erythritol phosphate pathway; 1-deoxy-D-xylulose-5-phosphate reductoisomerase Author: Carlota Roca, Yunuen Avalos-Padilla, Beatriz Prieto-Simón, Valentín Iglesias, Miriam Ramírez, Santiago Imperial and Xavier Fernàndez-Busquets Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Nov 23 08:34:32 2022 CET ModDate: Thu Nov 24 09:28:50 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 23 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5586185 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KORLVE+VnURWPalladioL Type 1 Custom yes yes no 10 0 NXQZWG+EURM10 Type 1 Builtin yes yes no 15 0 NYYLIC+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 RFTEAX+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 LXHBQN+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 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Destination: subsection.2.2 Item: Title: Subcloning and Sequencing of Candidate Oligonucleotides Destination: subsection.2.3 Item: Title: Computational Analysis Destination: subsection.2.4 Item: Title: Western Blots Destination: subsection.2.5 Item: Title: Electrophoretic Mobility Shift Assay Destination: subsection.2.6 Item: Title: Determination of Aptamer Dissociation Constants Destination: subsection.2.7 Item: Title: DXR Activity Assay Destination: subsection.2.8 Item: Title: P. falciparum Cultures Destination: subsection.2.9 Item: Title: Confocal Fluorescence Microscopy and Flow Cytometry Analysis Destination: subsection.2.10 Item: Title: Bacterial Cultures Destination: subsection.2.11 Item: Title: Ethics Statement Destination: subsection.2.12 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Aptamer Selection Destination: subsection.3.1 Item: Title: Characterization of Aptamer Binding to DXR In Vitro Destination: subsection.3.2 Item: Title: Characterization of Aptamer Binding to Plasmodium Cells Destination: subsection.3.3 Item: Title: Characterization of Aptamer Binding to Bacterial Cells Destination: subsection.3.4 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Selection of an Aptamer against the Enzyme 1-deoxy-D-xylulose-5-phosphate Reductoisomerase from Plasmodium falciparum Author: Carlota Roca, Yunuen Avalos-Padilla, Beatriz Prieto-Simón, Valentín Iglesias, Miriam Ramírez, Santiago Imperial and Xavier Fernàndez-Busquets Subject: The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 M a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naïve erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches. 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