TW201803991A - 用以篩選核酸適體之方法 - Google Patents

用以篩選核酸適體之方法 Download PDF

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TW201803991A
TW201803991A TW106102762A TW106102762A TW201803991A TW 201803991 A TW201803991 A TW 201803991A TW 106102762 A TW106102762 A TW 106102762A TW 106102762 A TW106102762 A TW 106102762A TW 201803991 A TW201803991 A TW 201803991A
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dna
selex
nucleic acid
aptamer
sequence
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吉本敬太郎
和久井幸二
古性均
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國立大學法人東京大學
日產化學工業股份有限公司
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    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2531/00Reactions of nucleic acids characterised by
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Abstract

本發明關於一種用以篩選核酸適體之方法,其包含下述步驟:(a)使固定於固相載體上之標的分子與核酸適體候選者接觸、(b)藉由毛細管電泳,分取與標的分子結合之核酸適體候選者、(c)藉由PCR而放大核酸適體候選者。

Description

用以篩選核酸適體之方法
本發明係關於用以篩選核酸適體之方法。
核酸適體係指具備分子辨識能力之單股DNA或RNA,於1990年由Ellington等人、與Tuerk等人首先報告。核酸適體可藉由稱為Systematic Evolution of Ligands by Exponential enrichment(SELEX)之進化工程學的方法獲得,亦被報告有多數之具有與抗體匹敵之結合能力或特異性者。進一步地,對於以蛋白質或細胞為首,不易取得抗體之低分子化合物等各種標的,能夠取得適體,期待對於治療藥或診斷藥之應用。但是,相對於抗體之獲得機率被認為有90%以上,目前核酸適體之獲得機率被認為係30%以下。換言之,核酸適體之獲得機率技術的提昇,可說是今後為了使核酸適體廣為於產業上利用的主要課題。
核酸適體係作為取代抗體之新穎的分子辨識單元而受到注目,但核酸適體之獲得率被認為係30%以下,期望高效率之適體獲得法的開發。CE-SELEX為利用 毛細管電泳之優良分離能力的核酸適體之迅速的篩選法。但是,由於有實驗條件設定(分取區域之設定)的難度、或作為標的之分子必須為與ssDNA庫結合時電泳移動度有大幅變化之一定以上的大小等限制,故通用性的缺乏係無法否認。
於CE-SELEX中,分取區域的設定,為左右適體獲得率的重要之要素。必須分取沒有混入對標的不具結合能力的ssDNA庫、且確實含有與標的形成複合體之ssDNA庫的區域。理想的分取區域,為來自ssDNA庫與標的之複合體的峰值範圍。但是,標的分子係如MutS蛋白質般,容易與ssDNA相互作用、且若不用高感度的螢光檢測器,則不易檢測ssDNA庫與標的之複合體。至今為止,有進行用以最佳化分取區域之各種設計,例如將標的蛋白質藉由螢光修飾予以可見化、或藉由即時定量PCR預測標的/適體複合體之檢測位置等。但是,複合體之檢測仍為困難,一般而言係將自標的分子之檢測位置起直到檢測到ssDNA庫之前作為分取區域。該分取區域之設定法,係有於途中解離之結合能力低的序列亦容易包含於分取區域中之問題點(圖1A)。
又,以低分子化合物等之小的分子為標的時,幾乎無法期待與ssDNA庫結合時之電泳移動度變化,因此被認為CE-SELEX之適用係困難的。藉由CE-SELEX而獲得對於低分子化合物之適體的例子,至目前為止僅有一例,由此亦可知其困難度(非專利文獻1)。
[先前技術文獻] [非專利文獻]
[非專利文獻1] Yang, J. & Bowser, M. T. Capillary Electrophoresis-SELEX Selection of Catalytic DNA Aptamers for a Small-Molecule Porphyrin Target. Anal. Chem. 85, 1525-1530 (2013)。
本發明提供在發揮CE-SELEX之長處的同時,解決了實驗條件設定之困難或可適用之標的分子種類少等缺點的新穎之CE-SELEX。具體而言,係關於組合了粒子與CE-SELEX的用以篩選核酸適體之方法。至今為止雖有報告組合了粒子與毛細管電泳之RNA或抗原的定量解析法,但組合了粒子與CE-SELEX之方法則尚未被報告。
本發明者等人深入研究之結果,發現藉由組