Systematic evolution of novel 2′F-PY RNA aptamers targeting the membrane protein l-arginine/agmatine antiporter purified in mild detergent

Ayoub, Nooraldeen
Bacterial microorganisms have developed numerous and diverse systems to handle potentially detrimental acidic conditionsintheir external environment. In particular, some pathogenic and other nonpathogenic enteric bacteriahave a number of exceptionaland well-organized acid resistance (AR) mechanismsthat work together to counter intracellular acidification and damage and enable survival under the extreme acidic conditions of the mammalian stomach. The common Escherichia coliwith both its virulent and benign strains is particularly remarkable considering itsadaptation to neutral pH. In these bacteria, the challenge of a low external pH is efficiently mitigated by highly potent proton consumption AR systems that consist of two generic components: a cytoplasmic pyrodoxal-5′-phoshpate (PLP)-dependent amino acid decarboxylase and an inner plasma membrane amino acid antiporter. Both,decarboxylation of a substrate amino acid into a product and CO2ina proton-dependent PLP-catalyzed manner and antiporter-mediated exchange of the internally generated decarboxylation products with the externally located substrate amino acid maintain the system running and aid in restoring pH homeostasis of the cell. One of these systems is the arginine-dependent acid resistance (ADAR) system constituted byacid-induced arginine decarboxylase (AdiA) andarginine-agmatine antiporter (AdiC). The impressive competence of AR systemsand especially those used by infectious, disease-causing enteric bacteria, comes at abigcost—the well-being and healthof humans. Henceforth, in this work, for the first time, weraisedfive candidate2′FY RNA candidate aptamers (noorA, noorB, noorC, AdiC19 and AdiC72) againstthe ADAR system’s integral membrane protein(MP), AdiC.Briefly, AdiC with a 10×Histidine tag ontheC-terminal wasrecombinantly overexpressedusing the convenient prokaryotic expression systeminBL21(DE3) pLysScell lineandwasextracted by solubilizingusing n-dodecyl-β-D-maltopyranoside(DDM) and purified by metal affinity chromatography in the same detergent. After confirming the puritybySDS-PAGE(sodium dodecyl sulphate polyacrylamide gel electrophoresis),recombinant AdiC was subjected to 8 rounds of conventional Systematic Evolution of Ligands by EXponential enrichment (SELEX)for the in vitroselection of2′F-Pyrimidine-modified candidate RNA aptamers. Oligonucleotide sequencesfrom the final library of selection werecloned and identified by Sanger sequencing. Three sequences (noorA noorB and noorC) were found abundant among 100 clones and thus chosen as the basis for subsequent informatics analyses. Using a computational approach withcomparative analyses of primary and optimal (MFE) 2D secondary structures predicted by RNAfoldand Kinefoldprograms, we were able to identify three conserved motifs (one of which we annotated as a putative local binding domain and the other two as stabilizers) in noorA, B and C and also in two more sequences(AdiC19 and AdiC72)identifiedina different phylogenetic family. We also defined aconsensus secondary structure to which thesefive proto-aptamer conform.Subsequent prediction-driven (in-silico) mutational deletions ofthestabilizer motifs revealed aglobal impact onseveral of the predicted MFE structures inmaintaining the substructure that harbors the putative binding motif. The five aptamer candidates generated in this work may have prospects as versatileagents applied inareas of aptamer-baseddiagnosticsand therapeutics where they maybe used to assessbacterial infections in patients(e.g. by aptasensors) or as antimicrobial pharmaceutical drugs or drug delivery agentsforthese infections. At the level of basic scientific research, these aptamers may also be useful in further understanding the structural features and biochemical functions of AdiC. More broadly, given the scarcity of SELEX trials against purified full-length MPs over the past ∼20 years, this thesis presents itself as one of the proof-of-principle studies that bring attention to the usability of purified full-length MPs for the selection of aptamers after MP reconstitution in detergent micelles. This advocacy is in line with the advantages provided by conventional protein-SELEX over cell-SELEX andutility ofsoluble intra-and extracellular MP domains usedastargets. At the end ofthis thesis, we direct the spotlightona special opportunity available forcomplex MPssuch asreceptors,transporters, andchannelsthat mayoften be inaccessible foraptamer targeting dueto multi-passing ordeep integrationto andintimacy with their membranes.


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Citation Formats
N. Ayoub, “Systematic evolution of novel 2′F-PY RNA aptamers targeting the membrane protein l-arginine/agmatine antiporter purified in mild detergent,” M.S. - Master of Science, Middle East Technical University, 2021.