Single-Molecule Biophysics

Single-Molecule Biophysics

Development now allows us to individual molecules at work. Conventional biochemistry experiments are restricted to the ensemble average. On the other hand, single-molecule experiments follow individual molecules going through reactions, and provide new and unique information that are averaged out in the conventional experiments.

Specifically, single-molecule experiments provide information about the reaction mechanisms and population distribution. Since individual molecules were followed through the reaction pathways, it is then possible to determine the reaction trajectories, and capture the short-lived, rarely populated reaction intermediates. In addition, by measuring many individual molecules, it is then possible to reconstruct the population distribution and compare it to the average values determined by conventional methods. This is like the census results are more informative than just the average income of the Taipei residents.

單分子生物物理

隨著影像技術的發展，科學家們已經有能力可以去操控、觀察單一分子的反應行為。一般傳統的生物化學實驗中，實驗數據受到大量分子數目平均的原因，使得許多有趣的反應過程，機制及分布，被隱藏在這群平均之下而無法觀察。而單分子實驗，則可藉由觀察「單一個」分子在化學反應進行的細節，藉此得到在傳統實驗中受到平均限制而無法觀測到的反應新資訊。

一般而言，單分子實驗可以提供我們對反應機制的進一步、更精細的觀察。分子發生反應時會遵循反應路徑進行，因此當我們觀察單一分子反應時，便有機會可以觀察到在一般平均實驗中很難觀察到的反應中間體（如存在時間很短或數目稀有的中間體），便可以藉此去提供反應機制的細節。此外，當我們觀察多個單分子反應，並累積足夠的數據時，即可利用統計方法去重建反應參數（如：速率常數、平衡常數、半生期……等）。單分子方法相較於傳統實驗方法可以提供更多細微的反應資訊，就如同做「人口普查」所得到的資訊會比直接只看「平均」獲得的資訊更多更有用。

Nano-Bioscience

We are developing new single-molecule experiments that can be used to study protein-DNA interactions at nanometer precision levels. Current approaches to visualize enzymatic action of individual biomolecules rely on monitoring the molecular probes attached to individual enzyme molecules or their substrates. Successful probes include polystyrene beads and quantum dots in sub-micron size. Even more powerful probes in single-biomolecule detection are individual fluorophores in sub-nanometer size. We are also developing a single-molecule optical tweezers manipulation platform to apply calibrated piconewton forces to a single enzyme-DNA complex.

Even though the diffraction limit of optical microscopy is about 200 nm, an image spot can in fact be determined with nanometer precision through digital image processing. For example, the image of a 100 nm polystyrene bead is a two-dimensional spot that varies in intensity and spreads across an area contains a number of pixels in the digitized image. The intensity profile of this image spot can be fit into a Gaussian curve that is a function of position x and y in the image. The position of the fitted Gaussian image, and hence the centroid position of the bead, determines the positional resolution of the bead centroid to a nanometer precision. It is thus possible to image individual biomolecules with nanometer precision.

奈米生物學

單分子領域的科學家們發展了不少新的單分子技術，可應用於奈米尺度下觀察單一蛋白質與DNA的交互作用，我們現在已經可以利用這些「分子探針」的技術觀察單一酵素分子的催化反應。例如利用次微米大小的乳膠小球連接到酵素本體或是受體上，利用觀測各個乳膠小球的位置進行實驗量測（單分子栓球實驗）。或是利用單一螢光分子直接標定在蛋白質或DNA上，直接觀察單一生化分子的構型變化或是結合情形（單分子螢光及螢光共振能量轉移）。我們也建立了「單分子光學鑷子平台」去施加特定大小（10^-12 牛頓）的力，對酵素-DNA複合體進行高精度的觀測（單分子光學鑷子）。