NANOSCALE: Elastin Nanobiosensors

这项研究的目的是通过将“软件”--一种为传感器编码的合成基因--插入细胞的“硬件”中，在细胞内创建一种基于蛋白质的分子传感器。 一旦启动该基因，细胞将产生功能性传感器分子，其将报告感兴趣的细胞内参数。 该传感器由两个集成组件组成：环境响应性弹性蛋白样多肽（ELP），可通过其氨基酸序列设计为对细胞内参数（如温度、pH或离子强度）非常敏感。 当细胞内参数发生变化时，ELP收缩，其端到端距离减少约50%。第二种组分是报告分子，其将ELP的这种纳米级收缩转化为读出信号：荧光的变化。 这是通过-蓝色荧光蛋白（BFP）-供体荧光团和-绿色荧光蛋白（GFP）-受体荧光团之间的荧光共振能量转移（FRET）来实现的，它们中的每一个在基因水平上融合到ELP的相对末端。 该建议的中心假设是ELP的崩溃，响应于感兴趣的细胞内参数的浓度改变，将改变供体（BFP）和受体（GFP）荧光团之间的距离，导致增强的FRET，从而提供可检测的荧光信号。 两种不同的生理传感器将被制造和测试：pH传感器，其中包括在ELP序列中的电离残基，和磷酸化传感器，其中包含在ELP内的肽底物。 改变的pH或激酶浓度将等温地诱导逆转变，导致由于改变的FRET的荧光读数。 这些原型传感器将在基础细胞和肿瘤生物学研究以及生物技术应用中找到应用，例如工业生物过程的实时细胞内监测。

The objective of this research is to create a protein-based molecular sensor within the cell by inserting the "software"-- a synthetic gene, which encodes for the sensor -- into the "hardware" of the cell. Uponturning the gene on, the cell will produce functional sensor molecules, which will report upon an intracellular parameter of interest. The sensor consists of two, integrated components: an environmentally-responsive, elastin-like polypeptide (ELP), which can be designed via its amino acidsequence to be acutely sensitive to intracellular parameters, such as temperature, pH or ionic strength. Upon a change in the intracellular parameter, the ELP contracts and its end-to-end distance decreases by ~50%.The second component is a reporter, which converts this nanometer scale contraction of the ELP into a readout signal: a change in fluorescence. This is achieved by fluorescence resonance energy transfer (FRET) between -blue fluorescent protein (BFP)- the donor fluorophore, and - green fluorescent protein (GFP) - the acceptor fluorophore, each of which are fused at the gene level to opposite ends of the ELP. The centralhypothesis of this proposal is that collapse of the ELP, in response to the altered concentration of an intracellular parameter of interest, will alter the distance between the donor (BFP) and acceptor (GFP) fluorophore, leading to enhanced FRET, and will thereby provide a detectable fluorescence signal. Two different physiological sensors will be fabricated and tested: a pH sensor, which incorporates ionizable residues in the ELP sequence, and a phosphorylation sensor, which contains a peptide substrate within the ELP. Altered pH or kinase concentration will isothermally induce the inverse transition, leading to fluorescence readout due to altered FRET. These prototype sensors will find application infundamental cell and tumor biology studies, as well as in biotechnology applications, such as real-time, intracellular monitoring of industrial bioprocesses.