Imaging Agent for Redox‐Active Molecules

Abstract

Redox‐active and cofactor molecules (such as glutathione, NAD+/NADH, and NADP+/NADPH) are involved in a wide array of important biological reactions. Their cellular concentrations are maintained within a range of values, and an imbalance is often linked to decreased cellular defense capability against oxidative stress. Currently, the most common techniques for quantifying these molecules require cell lysis. The goal of this project is to design sensor molecules that can be used in live cells. Our approach relies on the use of oligodeoxynucleotide strands (called aptamers) as the recognition moiety for the redox compound of interest. Aptamers, when selected for a specific target molecule, can display selectivity and affinity rivalling those of antibodies. Conjugation of the aptamer to a xenon‐containing organic molecule will yield an imaging agent that will report the concentration of the target as a measurable frequency value in the xenon nuclear magnetic resonance.Isolation of aptamers for a target molecule is achieved via an iterative in vitro selection technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Starting from a large pool of combinatorial library of aptamers (70‐base long strands containing 30 randomized bases), each iteration consists of: 1) incubation with the target molecule, 2) partitioning of the target‐binding and non‐binding aptamers, 3) amplification of the binding aptamers through polymerase chain reaction (PCR), and 4) separation of the amplicons into single‐stranded aptamers. This newly generated pool is then used as the starting library for the consecutive SELEX cycle. In the last round, the aptamer pool is cloned in E.coli and the sequence of each target binding strand is elucidated.The magnetic resonance frequency reporter is composed of 129Xe atom encapsulated in an organic ‘cage’ molecule. Starting from commercially available precursors, the synthesis of the Xe‐based component follows an established multi‐step organic synthesis procedure. In the final stage, conjugation of the aptamer to the organic ‘cage’ bearing the Xe atom is achieved via Huisgen 1,3‐dipolar cycloaddition or carboxylic acid‐amine condensation reaction.Toward this end, we have completed several rounds of SELEX and closely monitored the progress of each cycle. We started by incubating the aptamer pool with a high concentration of glutathione (10 mM), and observed aptamer binding to the target after several rounds. We have subsequently increased the selection pressure by gradually reducing the glutathione solution to 0.5 mM. At the same time, we have successfully completed several synthetic steps toward the Xe organic ‘cage’ molecule. The product of each step was fully characterized using standard organic chemistry methods.Upon completion of our project, we will have developed a magnetic resonance imaging (MRI) agent for glutathione which will be extended to other redox‐active and cofactor molecules.Support or Funding InformationFunding for the project came from the California State University East Bay (CSUEB) Faculty Support Grant. D.S. was a recipient of the CSUEB Center for Student Research Scholarship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.