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。入速率、出速率和平衡常数是工业和基础研究中广泛关注的问题。药物开发需要快速分离、识别和表征“强结合剂”小分子或生物制剂，特别是在筛选过程中。低温电镜激发了多组分蛋白质机器的研究，机械生物化学学科正在复苏。然而，蛋白质的生产和纯化是昂贵的，但为了生物真实性，必须在高浓度下使用，但大多数分析方法都会产生很大的稀释。这就产生了对小型化反应系统的需求，这些反应系统可以浓缩样品，允许精确操纵反应条件并进行分子表征。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过程中修饰另一种蛋白质，例如复合物的一些组分是酶，组装（或部分组装）复合物可能是酶，例如信号支架组件，核糖体，涂层，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纵生化相互作用的平台。凭借他们的原型QbQ（“立方”）系统，行业合作伙伴GMD建立了一个平台，可以在微流控通道中生成和操纵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蛋白质浓度、带组成、带宽度和带的运动方向。然后，我们将在穿越运动路径的意义上相互“碰撞”，而速度、浓度、相遇时间/持续时间和温度等特征则由软件控制。QbQ有潜力补充并超越最完善的分析方法。它避免了一个结合伙伴吸收到表面的空间位阻（SPR），并否定了质量传输限制（SPR和NMR）。QbQ保留了微尺度热泳（MST）、超动态光散射（DLS）和类似技术的优点，例如毫米长的光路和mg/ml浓度，但超过了MST，因为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。最终，这为逻辑操作和循环创造了可能性，以控制化学过程的进展。例如，“如果（样品A x样品B ==特征C），则C与D反应，否则C与D反应”。有了软件控制的化学反应，就有可能在一个小芯片上模拟复杂的、顺序的生化过程。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，但也会创建“价值分支点”，在这些分支点上，研究进展可以作为在其他应用（药物筛选）中有用的新设备的设计原则。这个项目符合BBSRC在DTP3申请中用于分配LIDo研究奖学金的定义，特别是“技术开发”。信号传导是BBSRC的普遍兴趣，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.