Evidence for rapid hydrolysis of shoot-derived sucrose using an ultrasensitive ratiometric Matryoshka-type MGlucoMeter sensor

To enable sensitive in vivo monitoring of the glucose transport and metabolism, we developed a series of ultrasensitive and ratiometric genetically encoded nanosensors by inserting a Matryoshka dual fluorophore cassette consisting of cpsfGFP and LSSmApple into the glucose binding protein ttGBP from Thermus thermophilus. The initial MGlucoMeter1.0 was subjected to an alanine scan of the hinge region producing more sensitive MGlucoMeter2.6 with a glucose-induced ΔF/F0 change of 3.0, an affinity for glucose of 15 µM, and an approximate detection range of 1.1-216 µM. To generate variants suitable for in vivo measurements, a series of affinity mutants was generated by mutating two histidines predicted to be involved in substrate binding. MGlucoMeter2.6-353n, MGlucoMeter2.6-15µ, MGlucoMeter2.6-700µ, MGlucoMeter2.6-1m, and MGlucoMeter2.6-7m cover a detection range between ∼40 nM - 55 mM. When expressed from a ubiquitous promoter in the cytosol of the Arabidopsis gene silencing mutant rdr6, MGlucoMeter2.6-1m reports time- and concentration-dependent accumulation of glucose in seedling roots. The sensor also detects rapid hydrolysis of shoot-derived sucrose in the root tip.

为实现对葡萄糖转运与代谢的高灵敏度活体监测，本研究将由cpsfGFP与LSSmApple组成的俄罗斯套娃式双荧光蛋白基因盒（Matryoshka dual fluorophore cassette）插入到来自嗜热栖热菌（Thermus thermophilus）的葡萄糖结合蛋白ttGBP中，开发出一系列超灵敏比率型基因编码纳米传感器（genetically encoded nanosensors）。初代MGlucoMeter1.0通过对其铰链区进行丙氨酸扫描诱变，获得了灵敏度更优的MGlucoMeter2.6：其葡萄糖诱导的ΔF/F0变化量为3.0，葡萄糖结合亲和力为15 μM，检测范围约为1.1~216 μM。为获得适用于活体检测的突变体，研究人员通过诱变预测参与底物结合的两个组氨酸残基，构建了一系列亲和力突变体。MGlucoMeter2.6-353n、MGlucoMeter2.6-15μ、MGlucoMeter2.6-700μ、MGlucoMeter2.6-1m以及MGlucoMeter2.6-7m的检测范围覆盖约40 nM至55 mM。将该传感器通过组成型启动子在拟南芥基因沉默突变体rdr6的细胞质中表达后，MGlucoMeter2.6-1m可实时反映幼苗根部葡萄糖随时间与浓度变化的积累过程。该传感器还可检测根尖处地上部来源的蔗糖的快速水解过程。