"Adaptation of tryptophan aptamer into an electrochemical aptamer-based sensor for use in tryptophan metabolism studies in the rat"

Yuyang Wu¹, Chelsea Brown², Zeki Duman³, Tod Kippin², Kevin Plaxco^1,*

¹Department of Chemistry and Biochemistry,
University of California Santa Barbara, Santa Barbara, CA 93106, USA
²Department of Psychological and Brain Sciences,
University of California Santa Barbara, Santa Barbara, CA 93106, USA
³Department of Electrical and Computer Engineering,
University of California Santa Barbara, Santa Barbara, CA 93106, USA

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. In this paper, we are engineering a tryptophan-detecting EAB sensor as tryptophan is an essential amino acid that is not only a building block for protein synthesis but also a precursor for the biosynthesis of co-enzymes and neuromodulators (NAD/NADP(H), kynurenic acid, melatonin and serotonin). Specifically, we used circular dichroism to identify a variant of an established tryptophan aptamer that undergoes a binding-induced conformational change for use in a tryptophan-detecting EAB sensor. We found that, while longer constructs (TRNC9/4, TRNC15/9) exhibit a significant change in ellipticity upon the addition of urea, a denaturant, the shorter constructs do not, suggesting that the former are folded in the absence of their target and the latter unfolded. When titrated with tryptophan and assayed by circular dichroism, an intermediate length construct, TRNC17/11, undergoes a significant conformational change upon target binding. Adapting this construct into an EAB sensor, we found it produces a large change in electrochemical signal at physiological tryptophan concentrations. The resulting device works well in 37 °C whole blood over the amino acid’s physiological range. Using this sensor, we obtained high-frequency, real-time plasma tryptophan measurements in vivo in live rats for tryptophan metabolism study in living rats. We found that, upon challenge with a single, high tryptophan dose, the resulting return-to-homeostasis kinetics are well-described by a Michaelis-Menton model limited by the Km of tryptophan dioxygenase. Upon a series of sequential tryptophan injections, the return-to-homeostasis rate accelerates, an observation that is consistent with reported literature that the activity of tryptophan dioxygenase in the liver is increased upon tryptophan challenge.

References:

1. Klaessens S, Stroobant V, De Plaen E, Van den Eynde BJ., Front Mol Biosci., 2022, 9, 897929.

2. Civen M, Knox WE., J Biol Chem., 1959, 234, 1787-1790.