A low velocity molecular collider for computer-controlled biochemical reactions.

研究生物分子相互作用有多种技术，如蛋白质-配体、蛋白质-核酸和蛋白质-蛋白质相互作用。开工率、开关率和平衡常数在工业和基础研究中具有广泛的意义。药物开发需要快速分离、鉴定和鉴定“强粘合剂”小分子或生物制品，特别是在筛选过程中。冷冻-EM为多组分蛋白质机器的研究注入了活力，机械生物化学学科正在复兴。然而，生产和纯化蛋白质的成本很高，但为了生物现实主义，必须在高浓度下使用蛋白质，但大多数分析方法会产生很大的稀释。这就产生了对小型化反应系统的需求，这些系统可以浓缩样品，允许精确控制反应条件，并提供分子表征。除了确定物理常数之外，还需要确定一种蛋白质是否在相互作用中修饰另一种蛋白质，例如，复合体的一些成分是酶，组装(或部分组装)的复合体可能是酶，例如信号支架组件、核糖体、辅酶、GroEL/ES伴侣等。我们将使用全新的电泳方法来创建一个计算机操作生化相互作用的平台。通过他们的原型QbQ(“立方体”)系统，行业合作伙伴GMD建立了一个平台，允许在微流体通道中产生和操作超尖锐分子带的运动(见下图1)。通过开发用于蛋白质工作的QbQ，我们将发现如何控制流体通道内的蛋白质浓度、条带组成、条带宽度和运动方向。然后，我们将在交叉运动路径的意义上使条带相互碰撞，而速度、浓度、相遇时间/持续时间和温度的特征由软件控制。QbQ具有补充和超越最成熟的分析方法的潜力。它消除了一个结合配对吸附到表面的空间位阻(SPR)，并消除了质量传输限制(SPR和核磁共振)。QbQ保留了微尺度热电泳法(MST)、超动态光散射(DLS)和类似技术的优点，如毫米长的光路和mg/ml的浓度，但由于稀释的蛋白质集中在芯片上，蛋白质的运动和相互作用可以同时控制，这最终为逻辑运算和循环控制化学过程的进展创造了可能。例如，“IF(样本A x样本B==特征C)则与D反应C，否则与C反应”。拥有软件控制的化学反应可以创造在一个小芯片中模拟复杂的、顺序的生化过程的可能性。该系统可用于定量(筛选)和定性(分析)调查。我们将专门研究信号转导中的蛋白质-蛋白质相互作用，但也将创建“价值分支点”，在这些分支点上，研究进展可以作为用于其他应用(药物筛选)的新设备的设计原则。该项目符合用于分配LIDO研究生院奖学金的DTP3应用程序中使用的BBSRC定义，特别是“技术开发”。信号转导是BBSRC的一个普遍兴趣，具体包括(I)再生生物学(Ii)免疫学和(Iii)干细胞目标。

There are diverse techniques for studying biomolecular interactions e.g. proteins-ligand, proteins-nucleic acidand protein-protein interactions. On-rates, off-rates and equilibrium constants are of wide interest in industrialand fundamental research. Drug development needs to quickly isolate, identify and characterise "strongbinder" small molecules or biologics, especially in screening. Cryo-EM has energised the study of multicomponentprotein machines and the disciplines of mechanistic biochemistry are being revived. However,proteins are expensive to produce and purify but for biological realism must be used at high concentrationbut most analytical approaches create large dilutions. This creates demand for miniaturised reaction systemsthat concentrate samples, allow precise manipulation of reactions conditions and give molecularcharacterisation. Beyond determination of physical constants there is also the need to establish if one proteinmodifies another during interaction e.g. some components of complexes are enzymes and assembled (orpartly assembled) complexes may be enzymes e.g. signalling scaffold assemblies, ribosomes, coatomers,GroEL/ES chaperones etc.We will use radically new electrophoretic methods to create a platform for computer manipulation ofbiochemical interactions. With their prototype QbQ ("cubic") system GMD, the industry partner, has built aplatform that allows the generation and manipulation of the motions of ultra-sharp molecular bands in amicrofluidic channel (See Figure 1 below). By developing QbQ for protein work we will discover how to controlprotein concentrations, band composition, band width and direction of motion of bands inside a fluid channel.We will then "collide" bands with each other in the sense of crossing motion paths while characteristics ofspeed, concentration, encounter time/duration and temperature are controlled by software.QbQ has potential to complement and then surpass the most well-established analytical approaches. Itavoids steric hindrance from absorption of one binding partner to a surface (SPR) and negates mass transportlimitations (SPR and NMR). QbQ preserves the advantages of microscale thermophoresis (MST) overdynamic light scattering (DLS) and similar techniques e.g. mm long light paths and mg/ml concentrations yetsurpasses MST because dilute proteins are concentrated on the chip and the motions and interactions ofproteins can be controlled simultaneously.Ultimately this creates the possibility for logical operations and loops to control the progression of chemicalprocesses. For example, "if (sample A x sample B == Characteristic C) then react C with D Else react C withE". Having software controlled chemical reactions can create possibilities of mimicking complex, sequentialbiochemical processes in a small chip. The system can lend itself to quantitative (screening) and qualitative(analytical) investigations. We will specifically investigate protein-protein interactions in signal transductionbut also create "value branch points" at which the research progress can be captured as design principles fornew devices useful in other applications (drug screening).This project fits the BBSRC definitions used in the DTP3 application for allocation of LIDo researchstudentships, specifically "Technology Development". Signalling is a general BBSRC interest and specificallyencompassed in (i) Regenerative Biology (ii) Immunology and (iii) Stem cells aims.