Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Summary

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

Abstract

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.

In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

Introduction

5'-Nucleoside triphosphates ((d)NTPs) represent a class of vital biomolecules that are involved in countless processes and functions ranging from being the universal currency of energy to regulators of cell metabolism. In addition to their role in these fundamental biological transformations, their modified counterparts have advanced as a versatile and mild platform for the introduction of functional groups into oligonucleotides, a methodology that nicely complements the automated solid-phase synthesis that is usually applied^1,2. Indeed, provided the (d)NTPs can act as substrates for RNA and DNA polymerases³, a wealth of functional groups inc....

Protocol

1. Synthesis of the Modified Nucleoside Triphosphates

The synthetic approach chosen follows the procedure developed by Ludwig and Eckstein since this method is generally reliable and leads to very few side-products (Figure 1A)³⁹.

1. Coevaporate the suitably 3’-OAc-protected nucleoside ( typically 0.1 mmol) twice with anhydrous pyridine (2 ml) and then dry under vacuum overnight. At the same time, dry tributylammonium pyrophosphate (0.13 mmol) under vacuum overnight.
2. Dissolve the nucleoside in a minimum of dry pyridine (0.2 ml) and add dry dioxane (0.4 ml) as a cosolvent. Finally....

Results

Modified nucleoside triphosphates are alluring synthetic targets since they allow for the facile introduction of an vast array of functional groups into nucleic acids⁴¹. However, the isolation and characterization of these activated building blocks is often revealed to be arduous. Consequently, the results shown herein are thought to provide a helping hand to follow the various steps within the aforementioned synthetic and biochemical procedures (Figure 1B).

In part.......

Discussion

The inclusion of modifications into nucleic acids is of interest for numerous practical applications including the development of antisense and antigene agents^42,43, labeling and functional tagging of oligonucleotides⁴¹, and in efforts to expand the genetic alphabet^44-46. Chemical alterations and functional groups are usually introduced into nucleic acids by application of standard and automated solid-phase synthesis protocols. However, the phosphoramidite building blocks need to be resil.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
γ-[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
Jupiter semipreparative RP-HPLC column (5μ C18 300 Å)	Phenomenex
Gene Q Thermal Cycler	Bioconcept	BYQ6042E
PCR vials	Bioconcept	3220-00
HPLC system	Amersham Pharmacia Biotech		Äkta basic 10/100
All oligonucleotides were purchased from Microsynth and purified by PAGE
5'-CAAGGACAAAATAC CTGTATTCCTT P1
5'-GACATCATGAGAGA CATCGCCTCTGGGCTA ATAGGACTACTTCTAAT CTGTAAGAGCAGATCC CTGGACAGGCAAGGAA TACAGGTATTTTGTCCTTG 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