Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Fluorescence Innovations Inc., Minneapolis, MN 55455, USA.
Biosensors (Basel). 2018 Oct 24;8(4):99. doi: 10.3390/bios8040099.
We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.
我们开发了具有红移荧光蛋白（FP）的荧光共振能量转移（FRET）生物传感器，从而提高了时间分辨（寿命）荧光测量的特性。与具有绿色和红色 FRET 对（GFP/RFP）的生物传感器相比，发射更长波长（橙色和栗色，OFP/MFP）的 FP 提高了 FRET 效率、动态范围和高通量筛选（HTS）的信号背景比。OFP 和 MFP 融合到人心脏钙泵（SERCA2a）的特定部位，用于检测小分子效应物引起的结构变化。当与最近改进的 HTS 荧光寿命微孔板读数器结合使用时，这种红移 FRET 生物传感器能够在三分钟的读取时间内，从微升样品体积中进行高精度纳秒分辨率的荧光衰减测量，每个 1536 孔板进行三次测量。使用小分子文库进行的初步筛选表明，OFP/MFP FRET 传感器可显著提高 HTS 测定的质量。这些高内涵 FRET 方法可以高精度地检测微小的 FRET 变化，这对于通过我们正在进行的 HTS 工作发现的小分子或肽调节剂来阐明新的结构机制非常重要。发射更长波长的 FRET 传感器对 FRET 生物传感器社区具有很大的吸引力，可用于药物发现和新治疗靶点的结构研究。
