定溫的方式探討Bcl2mid可逆控制G-四股展開與摺疊後的平衡差異

Abstract

富含鳥糞嘌呤 (G-rich) 序列可以藉由四個鳥糞嘌呤的鹼基經由Hoogsteen氫鍵可以形成一環狀的平面G-四方結構 (G-quartet)，由於G-tract的數量與鹼基多寡為G-四股結構添增了結構多樣性，以Bcl2mid (d[5’-G3CGCG3AGAAG5CG3-3’]) 為例，少一個G-tract會影響G-四股結構的形成，若其中一個G-tract含有五個連續的G序列，則易形成多聚體。此外G-四股結構的檢測不能單靠CD圖，需要搭配其他的實驗結果加以驗證，凡經過驗證後的CD圖則進一步追蹤四股結構之變化。 過去實驗室以加熱超過Bcl2mid的解旋溫度的方式，觀察到G-四股結構由單體轉換到雙體的熱力學產物，因此我們想藉由銅離子誘導G-四股結構的展開與EDTA抑制銅離子作用達到重新摺疊的方法，在定溫下探討G-四股結構的轉換。且在稀釋溶液 (dilute solution) 、20% PEG和20% Ficoll的不同環境條件下進行檢測，進而比較Cu/EDTA與退火 (anneal) 所形成的主要G-四股結構差異。我們運用CD光譜、非變性聚丙烯醯胺電泳、乳液誘導過濾來簡化系統的複雜度和模擬分子擁擠環境等驗證方法，來分析短時間生理溫度的環境下摺疊所得到的產物，實驗結果顯示其結構是以動力學產物為主。不同於將退火過程所產生的熱力學產物，在37˚C下做檢測，更為貼近生理條件下的結構轉換，有利於設計小分子對於動力學產物的篩選，有助於穩定生理溫度下G-四股結構的方法，達到抑制癌細胞生長。

Four guanine bases are held together by Hoogsteen hydrogen bonds to form a square planar G-quartet. Two or more quartets may stack on top of each other to form a G-quadruplex. The presence of G-quadruplex plays a significant role in potentially leading to the shortening of chromosome ends in the cancer cells and regulating gene expression. The number of G-tracts and the number of guanine bases in the G-tract gives a variety of structural morphology of G-quadruplex. For example, Bcl2mid (d[5’-G3CGCG3AGAAG5CG3-3’]). Missing a G-tract hinders the formation of G-quadruplex and preserving a G-tract with 5 consecutive guanine bases favors the formation of high order. Nevertheless, the most common apparatus for G-quadruplex examination, CD spectrum has been challenged for its reliability. Our experiment results show the inadequacy in G-quadruplex inspection for CD spectrum only while missing a G-tract; however, CD spectrum may apply on monitoring structural conversion of G-quadruplex once it has been confirmed on NMR spectrum. Previously our lab observed the structural conversion of Bcl2mid from a monomer to a thermodynamic product, dimer, upon heating above melting temperature; however, we would like to mimic the human body temperature and molecular crowding environment to study the possible structural conversion for Bcl2mid. We applied copper ion to induce the unfolding on G-quadruplex and EDTA2- chelates the copper ion to make random coil refolding back to G-quadruplex at a constant temperature. We have utilized CD, gel electrophoresis, emulsion induced filtration to study possible structural conversion. As a result, the kinetic product of G-quadruplex, monomer, in physiological temperature dominates in a short period of G-quadruplex refolding time. This finding may lead to a further studies on screening small molecules for the dominated G-quadruplex structure in the cells for the potential candidate of anti-cancer drugs.