核苷三磷酸 - 从合成到生化特征
Summary
本文所描述的协议旨在解释和删节的复杂路线导致修饰的三磷酸核苷的方法的许多障碍。因此，该协议有利于激活这些构建模块两者的合成及其可用性的实际应用。
Abstract
传统的策略用于引入化学官能团是通过附加适当修改的亚磷酰胺前体到新生链中使用的固相合成的。然而，合成和限制期间使用，以相当短的序列的条件妨碍本方法的适用性。在另一方面，修饰的核苷三磷酸激活已用于轻度引入众多的官能团为核酸，一种策略，铺平了道路，在实际应用广泛的调色板使用修饰的核酸构建模块如功能性标记核酶和脱氧核酶的产生。其中一个主要的挑战在于导致这些核苷类似物的分离和鉴定方法的复杂性。
在这个视频文章中，我们提出的成分股合成一个详细的协议用磷（Ⅲ）为基础的试剂ë修饰的类似物。此外，其生化特性的程序泄露，并特别强调对引物延伸反应和TDT拖尾聚合。这个详细的协议将用于修饰的dNTP的各具特色及其在化学生物学进一步利用。
Introduction
5'-三磷酸核苷（（四）国家结核病防治规划）是一类参与无数的流程和功能，从作为能源的通用货币为细胞代谢的调节是重要的生物分子。他们除了在这些基本的生物转化作用，其修改的同行拥有先进的引入官能团成寡核苷酸一种多用途的，温和的平台，一种方法，很好地补充了自动固相合成，通常是应用1,2。实际上，所提供的（D）的NTPs可作为RNA和DNA聚合酶3基板，具有丰富的官能团，包括氨基酸4-13，硼酸14,15，nornbornene 16，金刚石状残留物17，侧链为有机催化18，胆汁酸19，甚至20的寡核苷酸可被引入到寡核苷酸。
_content“>除了较方便的载体核酸的官能化，改性的dNTPs可从事SELEX和体外选择的用于改性催化核酸21-30和核酸适体用于各种实际应用10的产生等相关的组合方法， 31-36。被修饰的dNTP的聚合而引入的附加 侧链被认为是增加的化学空间，可在一个选择的实验探讨和补充的核酸37的相当差的功能的军火库。然而，尽管这些有吸引力的特征和近期的进展，合成和分析方法的发展作出了，没有普遍适用的和高产的过程存在修饰的核苷三磷酸2,38的各具特色。目前这个协议的目的是摆脱光线进入（有时）复杂的程序，导致吨Ø合成和激活这些积木（ 图1B）的生化特性。特别强调将在所有合成的细节，往往是很难找到或不存在的实验部分，但尚未至关重要，为圆满完成合成途径导致纯（四）国家结核病防治规划的隔离（ 图1）给出。
Protocol
1。修正的三磷酸核苷的合成选择的合成方法如下路德维希和埃克斯坦发展，因为这种方法一般是可靠的，并导致极少数的副产物（ 图1A）39的程序。 Coevaporate的适当3'-OAC-保护的核苷（一般为0.1毫摩尔）与两次无水吡啶（2ml）中，然后在真空下干燥过夜。在同一时间，干三丁基焦磷酸（0.13毫摩尔）在真空下过夜。 溶解核苷中的最小无水?…
Representative Results
修饰的核苷三磷酸是诱人合成的目标，因为它们允许轻便引入官能团的浩大到核酸41。然而，这些激活的构建块的分离和鉴定往往显露是艰巨的。因此，本文所示的结果被认为是提供一个帮手追随于上述合成和生物化学方法（ 图1B）中的各个步骤。 特别地， 图3示出了修改后的dNTP的典型粗31 P-NMR谱（在此特定情况下，dU的BPU <st…
Discussion
列入修改成核酸是利益的许多实际应用，包括反义和反基因药物的开发42,43，标签和寡核苷酸41的功能标记，并在努力扩大遗传字母44-46。化学改变和官能团通过应用标准和自动化固相合成用的协议，通常引入的核酸。然而，亚磷积木需要抵御由这种方法，这反过来又施加了严重的限制上的化学官能团47的性质所施加的相当苛刻的条件。相反，修饰的三磷酸核苷的酶…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由瑞士国家科学基金（批准N°PZ00P2_126430 / 1和PZ00P2_144595）的支持。 C. Leumann教授深表感谢提供实验室场地和设备，以及对他的一贯支持。苏克内希特女士是公认的富有成果的讨论。
Materials
| tributylammonium pyrophosphate | Sigma Aldrich | P8533 | Hygroscopic solid, keep under Ar |
| 2-chloro-1,3,2-benzodioxaphosphorin-4-one | Sigma Aldrich | 324124 | Moisture sensitive |
| Pyridine | Sigma Aldrich | 82704 | Under molecular sieves |
| Dioxane | Sigma Aldrich | 296309 | Under molecular sieves |
| dimethylformamide (DMF) | Sigma Aldrich | 40248 | Under molecular sieves |
| Acetonitrile | Fisher Scientific | HPLC grade | |
| Triethylamine | Sigma Aldrich | 90342 | |
| Tributylamine | Sigma Aldrich | 90781 | |
| ddH2O | Milli-Q | deionized and purified water, autoclaved in the presence of Diethylpyrocarbonate (DEPC) | |
| Diethylpyrocarbonate (DEPC) | Sigma Aldrich | 159220 | |
| D2O | Cambridge Isotope Laboratories, Inc. | DLM-4-25 | |
| Biochemical reagents | |||
| g-[32P]-ATP | Hartmann Analytics | FP-301 | |
| Natural dNTPs | Promega | U1420 | |
| Vent (exo–) DNA polymerase | NEB | M0257S | |
| DNA polymerase I, Large (Klenow) Fragment | NEB | MO210S | |
| 9°Nm DNA polymerase | NEB | MO260S | |
| Terminal deoxynucleotidyl Transferase (TdT) | Promega | M828A | |
| Pwo DNA polymerase | Peqlab | 01 01 5010 | |
| T4 PNK | Thermo Scientific | EK0032 | |
| Acrylamide/bisacrylamide (19:1, 40%) | Serva | 10679.01 | |
| Agarose | Apollo Scientific | BIA1177 | |
| G10 Sephadex | Sigma | G10120 | |
| Urea | Apollo Scientific | BIU4110 | |
| Equipment | |||
| Jupiter semi-preparative RP-HPLC column (5m C18 300Å) | Phenomenex | ||
| Gene Q Thermal Cycler | Bioconcept | BYQ6042E | |
| PCR vials | Bioconcept | 3220-00 | |
| HPLC system | Amersham Pharmacia Biotech | Äkta basic 10/100 | |
| Oligonucleotides | |||
| All oligonucleotides were purchased from Microsynth and purified by PAGE | |||
| 5'-CAAGGACAAAATACCTGTATTCCTT P1 | |||
| 5'-GACATCATGAGAGACATCGCCTCTGGGCTAAT-AGGACTACTTCTAATCTGTAAGAGCAGATCCCTGG-ACAGGCAAGGAATACAGGTATTTTGTCCTTG T1 | |||
| 5'-GAATTCGATATCAAG P2 | |||
| More information on experimental procedures and equipment can be found in the following articles: | |||
| Chem. Eur. J. 2012, 18, 13320 – 13330 | |||
| Org. Biomol. Chem. 2013, DOI: 10.1039/C3OB40842F. |
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Cite This Article
Hollenstein, M., Smith, C. C., Räz, M. Nucleoside Triphosphates – From Synthesis to Biochemical Characterization. J. Vis. Exp. (86), e51385, doi:10.3791/51385 (2014).