Construction Of A Genetically Encoded NADH Fluorescent Probe Based On Thioredoxin System

Nicotinamide adenine dinucleotide(NADH)is an imperative coenzyme and core metabolite in life system.It involves in redox reactions and plays vital roles in various cellular metabolic pathways and energy transfer processes such as glycolysis and tricarboxylic acid cycle.The main source of NADH includes the glycolysis and tricarboxylic acid that are carried out in cytoplasm and mitochondria,respectively,and the fluctuation in concentration of NADH indicates the intracellular metabolic states.Therefore,the regulation of the intracellular metabolic pathways and the physiological states of the cells can be well represented by monitoring the dynamic changes of intracellular NADH in real-time.The Thermoplasma acidophilum thioredoxin system in archaeon contains of Thioredoxin(Ta-Trx)and Thioredoxin reductase(Ta-TrxR).Ta-TrxR is a flavin protein,its main role is to reduce oxidized Ta-Trx via the electron transfer by NADH followed by reduction of intracellular disulfide-containing target proteins.The reduced Ta-Trx regulates several physiological and biochemical reactions in the organism.Based on the thioredoxin system of the Thermoplasma acidophilum,we developed a new NADHspecific genetically encoded fluorescent probe.Here,a protein conjugate TrxRFP was formed by coalescing Ta-Trx with a redoxsensitive red fluorescent protein(rxRFP)via a polypeptide.This was realized by coexpression of their coding sequences in HEK(Human embryonic kidney)293T cells.When the intracellular NADH level is elevated,the electrons of NADH are transferred to the oxidized Ta-Trx in the presence of Ta-TrxR.The reduced Ta-Trx further reduces the disulfide bond in rxRFP and the redox characteristics of rxRFP changes from the oxidized state to the reduced state,resulting in significant reduction of fluorescence intensity.Therefore,we can determine the dynamic change of intracellular NADH by detecting change of red fluorescence intensity.Characters such as the specificity,response time,sensitivity and dynamic changes of the probe were evaluated in HEK293 T cells.In this project,we constructed a new NADH-specific genetically encoded fluorescent probe and evaluated its applicability in cells.We have tried the application of thioredoxin system of thermoplasma acidophilum to design genetically encoded fluorescent probes,providing a new idea and direction for the development of diversified genetically encoded fluorescent probes in future.