利用单分子操控术进行DNA奈米剂量之创新研究

1、利用單分子操控術進行DNA奈米劑量之創新研究許志楧 吳見明 樊台清 董傳中 江國寧 江啟勳 洪志宏 羅建苗 許文郁 許靖涵根據醫學統計，人類每三到四人中就有一人在其有生之年罹患癌症。而輻射治療為治療癌症的主要方法之一。輻射治療劑量學從早期之組織層級的巨觀之劑量(dosimetry)到一、二十年前開始進入細胞層級的微劑量(microdosimetry)，這一、兩年來以DNA分子為主要探討標的奈米劑量(nanodosimetry)則剛萌芽。在從事輻射治療的過程中，輻射劑量在治療部位的分佈之計算評估(專業術語為：輻射治療計畫Treatment Plan)，為輻射治療最關鍵的工作之一。因為唯有精確的劑

2、量評估才能達到有效的殺死癌細胞並且對正常細胞/組織的殺傷力降到最低。這就像對某種疾病我們已研發出非常有效的葯物，但是若不知正確的使用劑量，則可能不是用量太低而無效；就是用量太重而毒性太高。而輻射劑量的評估是一個非常複雜的三度空間及時間的問題，它亦涵蓋了物理、化學、生物醫學的知識。要達到精確的輻射劑量評估，就要有精確的輻射劑量物理模式(Model)，而要驗證及修改所建構的輻射劑量物理模式則需要有實驗相互輔助。從整個巨觀之劑量學到微劑量的發展都是如此的，以模式和實驗交互成長而來。而輻射對細胞的殺害主要是打斷DNA分子(2nm寬)的作用。故要精確的評估輻射劑量必然有賴於正確的奈米劑量物理模式的建立。

3、而單分子操控技術則提供了最直接的實驗數據來驗證及修改所建立的奈米劑量物理模式。而其最終的正確性則需要輻射生物/醫學的驗證(子計畫4)。奈米劑量對輻射治療的重要性可以由目前的一個臨床經驗看出。肝癌一直被認為很難由輻射治療來治癒。縱然使用像粒子這種高能量轉移的輻射亦然。其原因乃在於，像粒子這種高能量轉移的劑量，無法由現今使用的劑量學正確的估算。因此，導致臨床上對這於將這種高效能的輻射，用之於治療肝癌產生卻步，因而無法有效的予以治癒。另外，美國的Lama Linda University為全球輻射治療的先驅之一。他們是第一個在University Hospital建置質子加速器進行質子輻射治療的大學

4、。有鑑於奈米劑量研究的重要性，他們在兩年前即結合了美國西岸四個大學/研究單位(LLU, WIS, UCSD, UCSC) 的物理、化學、生物醫學領域的研究人員，啟動了一個五年研究計畫，進行奈米劑量之研究。值得一提的是該研究計畫使用的仍然是傳統的微劑量的縮小化(Scale Down)，而沒有用到單分子操控術。所以，我們若能得到支助，趕快開始此一研究，我們在這方面的突破與領先仍是可期的。另外，值得一提的是，若將來以輻射照射處理奈米元件或是將奈米元件送到外太空等，輻射在奈米尺度的作用機制的了解都將提供很有用的知識。如前所述，奈米劑量本身即為一非常前衍的研究領域。而該研究的主要困難之一為實驗的驗証。目

5、前尚無以奈米技術進行這類實驗的文獻報告。本研究計畫針對此一極為前衍之問題，結合了奈米生技之單分子操控術，提出了一完全創新的奈米劑量實驗研究：利用光鑷子操控單一的雙鏈DNA分子，並結合分子影像技術中輻射藥物分子的輸送與標誌DNA分子(子計畫5)，來達到奈米劑量的施予。在這些施放能量作用有效尺度為奈米等級(正好是雙鏈DNA的橫向尺度等級)的放射性分子的作用下，DNA的單鏈斷裂或是雙鏈斷裂則由光鑷子拉力之改變量測得到(子計畫1)。配合實驗，國內劑量學的權威董教授將進行奈米劑量的理論推導與模型建構(子計畫2)，DNA拉力行為的模擬研究(子計畫3)亦包含在計劃中。經過理論與實驗的相互引導、印證，應能對此

6、一主題有較全面性的了解。由於以單分子技術進行這樣的實驗是完全創新的，所以除了與理論預期結果的對照之外，我們亦將進行一些多分子與細胞層級的生化實驗來對部份單分子的實驗結果，進行驗証(子計畫4)。更進一步，利用奈米劑量實驗技術的發展，我們將研究其應用於特定基因分子輻射治療之可能性。由於有臨床醫師的參與，這樣的應用研究將更為實際。單分子的實驗數據經常隱藏在大量的雜訊之中。因此，在本研究中的實驗數據分析與影像分析將因為有統計與影像的專長人員的參與(子計畫6)而得到很大的助益。各子計畫之間的關聯性及相互支援的密切關係，請參閱位於英文摘要的示意圖。另外，各計畫時程上的相互支援關係及檢測點(Checking

7、 points)，則請參閱A4. Project Description末之圖示。在西元2000年4月美國國衛院NIH(NIGMS)舉行了一個有關單分子偵測與操控的研討會。會中大家覺得，這一領域的奈米生技生醫非常重要 (htt gov/news/meetings/single_molecules.html#content)。因此，在西元2001年2月，美國國衛院就針對單分子偵測與操控公告徵求計畫書 files/ PA-01-049.html)。依據其計畫資料庫(CRISP)顯示，在西元2001年及2002年美國國衛院共支助了276個單分子偵測與操控的相關研究計畫(其中多數為多年期計畫)。在所有

8、這些單分子偵測與操控的相關研究計畫中，尚無任何計畫是利用單分子技術來進行奈米劑量之研究的。本研究團隊的特色是真正的跨領域團隊(生技、物理、光電、機械、生物、醫學、化學、統計、電機)，而且是針對一個單一主題的各個面向的需求而密切的結合在一起。除了第二、三子計畫的co-PIs及洪教授外，所有的co-PI都是每個星期，一起召開Group Meeting或是一起參加Journal Club，此一大型研究團隊的成員，早已建立了良好的合作關係與配合默契。本研究團隊的成員不管是在理論劑量學、放射藥物標誌、輻射生物學都是國內的一時之選，單分子操作術則得到史丹佛大學朱隸文教授及Steve Block教授的協助。

9、本計畫總主持人從事加速器物理複雜系統研究及光電研究多年，近年致力於將其實驗方法應用於生命科學研究，透過和史丹佛大學醫學院之合作，成功的在清華大學建立起以基因晶片系統為基礎的分子生醫光電實驗室。這樣密切結合的完整跨領域團隊配合持續有效的國際合作資源，在奈米劑量的國際領域中，應該是非常具有競爭力的一個組合(請參閱本節末附圖一)。如此有競爭力的研究團隊加上創新的想法與積極的跨領域做法，給我們充份的支持，三、五年內在奈米劑量的領域要成為國際一流的卓越團隊，我們應該有很好的機會。DNA Nanodosimetry Novel Study by Single Molecule Techniques許志楧

10、吳見明 樊台清 董傳中 江國寧 江啟勳 洪志宏 羅建苗 許文郁 許靖涵Gene-specific molecular radiotherapy, combining the features of high RBE (relative biologic effectiveness) of radiotherapy and specific gene targeting of gene therapy, has promised to be a new evolution in cancer therapy. This new type of techniques relies on the s

11、uccess of the development of sequence-specific DNA cleavage by a cascade of low-energy electrons production through an Auger electron emitter (A-Ettr). The idea of using an A-Ettr-labeled oligonucleotides as radiopharmaceuticals has become more promised in last few years. However, it has not been ad

12、apted into clinics. The main limitation is the radiation dose that occurs in nanometer range cant be determined by any current methods. The development of a dosimetry that can measure the radiation events in nanometer scale will be a key to the maturation of this technique. The overall aim of this p

13、roject is to develop a novel DNA nanodosimetry by single molecule techniques. The traditional evaluation of radiation biological effects for internally deposited radionuclides is in terms of the absorbed dose to an organ of interest. The implicit assumption based on this evaluation is that the indiv

14、idual cell dose in an organ is equal to the average organ dose, i.e. the radiation dose is homogeneously distributed over the whole organ. Such an assumption is adequate on the condition that the emitted radiation (e.g. photons or energetic electrons) has a range much longer than the cellular dimens

15、ions. In this case, the overwhelming contribution to the individual cell dose results from radioactive decays outside of the cell, regardless of their exact origin (whether extracellular or intracellular). When the emitted radiation (e.g. alpha particles or low energy electrons) has a range comparab

16、le to or smaller than the cellular dimensions, the exact location of radionuclides in or near the cell becomes significant. This is due to the highly localized energy deposition of short ranged particles in the immediate vicinity of the decay site. Since radiosensitive targets, for instance DNA, are

17、 presumably located in the cell nucleus, the position of the radionuclides relative to these targets is thus relevant. Therefore, the primary interest in evaluating radiation biological effects in nuclear medicine, radiation therapy and radiation protection shifts from macroscopic organ dose (dosime

18、try) to microscopic cell dose (microdosimetry), then further to mesoscopic DNA dose (nanodosimetry). The importance of the nanodosimetry on the success of radiotherapy can be highlighted by following clinical example. Liver cancers are generally recognized as difficult to be cured by radiotherapy, e

19、ven by high LET (linear energy transfer) radiation, such as a particles. Since the radiation dose of short ranged particles such as a particles can not be accurately measured by current dosimetry, clinicians therefore hesitate to deliver enough radiation doses and consequently result in treatment fa

20、ilure.The biological influence of short ranged particles, especially of Auger electrons, in human cells is the main focus of research activities on the action of ionizing radiation. Since the great majority of Auger electrons have energies less than 1 keV and often are emitted in showers of tens of

21、electrons per decays, they deposit radiation energy with very high concentration within a radius of several nanometers. The RBE for Auger emitters depends critically on the location of the radioisotope in human cells. For emitters such as I-125, located inside a cell but outside the DNA, RBE values

22、up to 8 have been observed. Further, for I-125 incorporated into DNA, RBE values of 20-40 have been found based on cell transformation. Thus, the establishment and the development of a nonodosimetry model for Auger electrons are important and necessary for dose estimations in medical application of

23、Auger electron emitters. This project aims to develop a novel DNA nanodosimetry by single molecule techniques. We will measure the breakdown of single and double strand of a single DNA molecule by optical tweezers and smFRET (single molecule Fluorescence Resonance Energy Transfer). This type of appr

24、oach is novel and has not been used previously. Technology wise, Optical Tweezers has been developed for almost one decade. There are commercially available Optical Tweezers systems. However, up to now, all of the commercial Optical Tweezers systems are mainly used for control the motion and positio

25、n of cells and other similar objects. Only up to a couple years ago, nanometer range resolution Optical Tweezers systems with the capability of measuring the picoNewton force are being constructed in several world frontier research laboratories, e.g. Block Lab at Stanford University, USA and Yanagid

26、a Lab at Japan. SmFRET techniques dont have commercial systems, neither. Examples of the world frontier research laboratories are Chu Lab at Stanford, and Weiss Lab at Berlekey. Applying the above techniques into the nanodosimetry study has its high threshold because one needs to gather Optical Twee

27、zers system construction and experiments PIs, nanodosimetry theoreticians, radiopharmaceutical experts, and radiation biologists. And, they have to work together very closely. Those are actually advantages of our team. By the way, the PI of this proposal, Prof. Hsu, has contacted with Prof. Block an

28、d Prof. Chu and both of them have agreed to serve as the members of the advisory committee of the Single Molecule Core Lab, which was proposed by us and other universitys faculty in UST (University System of Taiwan). Some of PIs and students of this proposal have been visited and discussed with both

29、 of them and obtained useful experience which are very helpful to the simplified Optical Tweezers system which we are constructing now. In April 2000, US NIH(NIGMS) held an workshop, NIGMS Single Molecule Detection and Manipulation Workshop April 17-18, 2000. news/meetings/single_molecules.html#cont

30、ent). T pa-files/PA-01-049.html) of SINGLE MOLECULE DETECTION AND MANIPULATION at Feb. 2001 by NHGRI, NIGMS, and NIDCD. Using the CRISP of NIH (Computer Retrieval of Information on Scientific Projects), by typing Single Molecule for searching, we found there are 276 single molecule projects funded b

31、y NIH in years 2001 and 2002(most of them are continues projects). It shows the importance of the single molecule research in the bio-nanotechnology. However, among all of those single molecule related projects, none of them is utilizing the single molecule techniques to study the nanodosimetry, yet

32、. The indication is that the idea of using single molecule technique for the study of nanodosimetry is novel. It may also imply the difficulty for doing this type of research. In other words, this type of research is difficult to be accomplished in a single laboratory. It requires a cooperation of s

33、pecialists from different areas, such as dosimetry, photonics, molecular biology, and radiopharmaceuticals. The importance of interdisciplinary approach is easy to appreciate, but its operation is difficult. It order to achieve such goal, we have organized an interdisciplinary team. The interaction

34、and cooperation of each member in this team is our strength and the key to the success of this project. Our approach to nanodosimetry is by two directions, theoretical modeling and experimental working. The main technique that we will use is optical twizzer and smFRET (component project 1). The expe

35、rimental results will be used to modify the model presented in component project 2 and 3. On the other hand, the model proposed by component project 2 and 3 will be the base for experimental design in component project 1 and 4. The mutual interaction between modeling and experimental will lead us to the development of a best nanodosimetry. Since the approach by single molecul