Saran Dayal, Burke Donald H
Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
Bioconjug Chem. 2007 Jan-Feb;18(1):275-9. doi: 10.1021/bc060221f.
Covalent photocleavable attachment of small molecules or peptides to oligonucleotides is an integral strategic element in the selection of novel nucleic acid enzymes. Here, we report the synthesis of a multipurpose, photocleavable bifunctional linker (PCBL) suitable for nucleic acid selections and other biotechnology applications. PCBL contains a photocleavable O-nitrobenzyl group flanked on one side by an N-hydroxysuccinimidyl ester (reactive toward primary amines) and on the other side by a sulfhydryl. To demonstrate the utility of PCBL, the linker was used to couple an analog of the antibiotic chloramphenicol (Cam) to the 5' end of an amino-modified 8-mer DNA oligo. Coupling was confirmed by MALDI-TOF spectrophotometry. Decoupling was performed by irradiating the coupled species with near-UV light (approximately 360 nm), regenerating the original amino-modified oligo. Ligation of the Cam-PCBL-DNA conjugate to random-sequence RNA generated a diversity library appropriate for the selection of new ribozymes that catalyze reactions involving the tethered substrate. Coupling and decoupling of the Cam analog from the library was monitored on a trilayered organomercurial polyacrylamide gel. The coupling/decoupling strategy described here is readily generalized to many combinations of macromolecules and small molecules. For example, analogs of this small molecule-DNA conjugate can be generated as synthons for ligation to nucleic acid diversity libraries during each round of novel ribozyme selections, or they can be immobilized onto chips for addresssably reversible microarray analysis.
小分子或肽与寡核苷酸的共价光可裂解连接是新型核酸酶筛选中不可或缺的战略要素。在此，我们报告了一种适用于核酸筛选及其他生物技术应用的多功能光可裂解双功能连接子（PCBL）的合成。PCBL含有一个光可裂解的邻硝基苄基，一侧为N - 羟基琥珀酰亚胺酯（对伯胺有反应活性），另一侧为巯基。为证明PCBL的实用性，该连接子用于将抗生素氯霉素（Cam）的类似物偶联到氨基修饰的8聚体DNA寡核苷酸的5'端。通过基质辅助激光解吸电离飞行时间质谱法确认了偶联。通过用近紫外光（约360 nm）照射偶联物进行解偶联，再生出原始的氨基修饰寡核苷酸。将Cam - PCBL - DNA缀合物连接到随机序列RNA上产生了一个适用于筛选催化涉及连接底物反应的新核酶的多样性文库。在三层有机汞聚丙烯酰胺凝胶上监测Cam类似物与文库的偶联和解偶联。此处描述的偶联/解偶联策略很容易推广到许多大分子和小分子的组合。例如，这种小分子 - DNA缀合物的类似物可以作为合成子生成，用于在每轮新型核酶筛选期间连接到核酸多样性文库，或者它们可以固定在芯片上用于可寻址的可逆微阵列分析。
