Phosphoramidation反應之應用於核酸與胜肽、蛋白質、螢光分子、或生物素衍生物鍵結生成的探討

本研究中，首先經由設計合成出生物素衍生物6-[5-(2-Oxo -hexahydro-thieno[3,4-d]imidazol-4-yl)-oentanoylamino]-hexanoic acid[6-(2-amino-acetylamino)-hexyl]-amide (11)，並利用11進行phosphoramidation反應，開發出新的且更有效的核酸與受質鍵結方法，以期利用於篩選具有催化水解醯胺鍵活性的核酸酶。由於現有phosphoramidation反應方法無法使核酸-11鍵結產物達到較高的產率，因此我們藉著改變實驗流程及反應條件，完成phosphoramidation反應的最佳化，這個新的phosphoramidation反應，在反應3小時後，就能使核酸-11的鍵結產物，達到80%的產率。很遺憾在利用該方法活性篩選完RNA池後，我們沒有發現到任何具有催化水解醯胺鍵活性的核酸酶，但我們後續實驗證實，該新穎的phosphoramidation反應，可以運用在核酸與任何含有胺基的分子間的鍵結，這些分子可以是生物素衍生物、螢光劑、蛋白質、或是胜肽。我們實驗結果也證明經由適當的控制phosphoramidation反應，我們能使核酸的鹼基配對特性產生最小的傷害，有效的製備出核酸與胜肽鍵結產物 (peptide-oligonucleotide conjugates, POCs），這些經由phosphoramidation反應合成出的POCs產物，可以在未來應用於RNA干擾(RNA interference)、基因沉默化(gene silencing)、及基因治療(gene therapy)等，開發出具有細胞專一性且更具療效的POCs藥物，或是運用於其他基礎科學研究。

關鍵字

胜肽蛋白質

並列摘要

The original aim of this research was to develop a new phosphoramidation reaction suitable for nucleic acid and small organic compound conjugations which could be exploited to identify amidase ribozymes from RNA pools. An amine-containing biotin derivative (11) was synthesized and used to systematically optimize a previously ineffective and inefficient phosphoramidation reaction method. The improved phosphoramidation reaction increased yields of nucleic acid-11 conjugates up to 80% after 3-hr reactions. Applications of the phosphoramidation reactions to conjugate 96 RNA clones with 11 and studies of the enzymatic activities for these RNA-11 conjugates revealed that all RNA conjugates had no detectable amidase activity. We, however, demonstrated any nucleic acids with a terminal phosphate group were suitable reactants in the new phosphoramidation reactions to conjugate with nucleophile molecules such as derivatives of biotin and fluorescein, proteins and peptides. Polymerase chain reaction (PCR) studies of 11- or fluorescein-tagged DNA primers prepared by the phosphoramidation reactions indicated base-pairing characteristics of the nucleic acid conjugates were substained. This study developed a facile phosphoramidation approach to prepare nucleic acid conjugates with good yields which have implications for broad biomedical applications.