Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 including amino acids^4-13, boronic acids^14,15, nornbornene¹⁶, diamondoid-like residues¹⁷, side-chains for organocatalysis¹⁸, bile acids¹⁹, and even oligonucleotides²⁰ can be introduced into oligonucleotides.

Beyond representing a convenient vector for the functionalization of nucleic acids, modified dNTPs can be engaged in SELEX and other related combinatorial methods of in vitro selection for the generation of modified catalytic nucleic acids^21-30 and aptamers for various practical applications^10,31-36. The additional side-chains that are introduced by the polymerization of the modified dNTPs are thought to increase the chemical space that can be explored during a selection experiment and supplement the rather poor functional arsenal of nucleic acids³⁷. However, despite these attractive traits and the recent progress made in the development of both synthetic and analytical methods, no universally applicable and high-yielding procedure exists for the crafting of modified nucleoside triphosphates^2,38.

The aim of this present protocol is to shed light into the (sometimes) intricate procedures leading to the synthesis and biochemical characterization of these activated building blocks (Figure 1B). Special emphasis will be given on all the synthetic details that often are difficult to find or are absent in experimental sections but are yet crucial for the successful completion of the synthetic pathway leading to the isolation of pure (d)NTPs (Figure 1).

Protokół

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, add 2-chloro-1,3,2-benzodioxaphosphorin-4-one (0.11 mmol) and allow to react at room temperature for 45 min.
   Note: It is noteworthy that particular care should be taken with the 2-chloro-1,3,2-benzodioxaphosphorin-4-one reagent. Indeed, even storage of this reagent under an inert gas atmosphere is not sufficient to prevent its decomposition. Thus, the white solid that is formed in this decomposition can and should be scraped off prior to use.
3. Prepare a solution of tributylammonium pyrophosphate in dry DMF (0.17 ml) and freshly distilled tributylamine (58 µl; never add molecular sieves). Add the resulting solution to the reaction mixture (a white precipitate appears but quickly disappears) and allow to react at room temperature for 45 min.
4. Prepare a solution of iodine (0.16 mmol) in pyridine (0.98 ml) and H₂O (20 µl) and add to the reaction mixture in order to oxidize the Pα(III) center. Allow the resulting dark solution to stir at room temperature for 30 min.
5. Use a 10% aqueous solution of NaS₂O₃ to quench the excess I₂. Remove the solvent under vacuum (the temperature of the water bath of the rotary evaporator must be kept below 30 °C). Add H₂O (5 ml) and allow the mixture to stand at room temperature for 30 min to hydrolyze the cyclic triphosphate moiety.
6. At this stage, the protecting groups are usually removed (i.e. the 3'-OAc and the groups on the side chains of the nucleobase). Consequently, add NH₄OH (30% in H₂O, 10 ml) to the crude and stir for 1.5 hr at room temperature, and remove the solvent under vacuum.
7. Add 2 ml of H₂O and split into 2 tubes. Add 12 ml of a 2% solution of NaClO₄ in acetone and centrifuge (1,000 x g) for 30 min. This procedure is repeated one more time. This precipitation allows for the separation of the solvents and reagents used in the synthesis from the triphosphate (which will precipitate), thus simplifying the ensuing RP-HPLC purification substantially.
8. After air-drying of the oily residue, record a ³¹P-NMR spectrum of the crude (following standard procedures, see also List of Materials and Figure 3) and dissolve in 4 ml H₂O.
   Note: This synthetic procedure mainly focuses on the generation of modified deoxynucleoside triphosphates, but a very similar procedure can be applied for their RNA counterparts (by simply using 2',3'-bis-O-acetylated precursors).

2. HPLC Purification of the Modified Nucleoside Triphosphates

1. Preparation of a 1 M stock solution of triethylammonium bicarbonate (TEAB):⁴⁰
   1. Prepare a solution of 1 mole of triethylamine (139 ml) in ≈ 600 ml of distilled and filtered H₂O.
   2. Bubble CO₂ (via dry ice and a gas bubbler) into the solution under vigorous stirring until a pH of 7.6 is reached (this will take at least 10 hr). This stock solution can be stored in the fridge for up to one month.
2. Purification:
   1. Prepare two buffer solutions from the 1 M stock: 2 L of 50 mM TEAB in ultrapure water (eluent A) and 1 L of 50 mM TEAB in 50% MeCN (eluent B). Degas both eluents under vacuum and stirring for 20 min (careful attention is required so as not to alter the pH by removing CO₂ during degassing).
   2. Prepare an analytical sample by dissolving 10 µl of the crude triphosphate in 300 µl distilled H₂O and inject into an HPLC system equipped with a semi-preparative RP column (C18) and using a gradient ranging from 0-100% B in 40 min (Figure 4). Adjust the HPLC program according to the R_t of the triphosphate (which is usually the main peak on the chromatogram) and the diphosphate (which has a slightly lower R_t). Purify the crude mixture using these conditions and by removing early fractions that might contain some diphosphate.
   3. Combine all the fractions that contain the product and freeze-dry (which is preferred to evaporation on the rotary evaporator in order to minimize hydrolysis to the undesired diphosphate). Coevaporate several times with ultrapure water (to remove remnant triethylamine from the eluents). Assess the purity of the triphosphate by NMR (both ¹H and ³¹P NMR) and MALDI-TOF by application of standard protocols (see Figure 5). Typical yields obtained by application of this protocol typically lie in the range of 30-70%, depending on the substrate.

3. Primer Extension Reactions and TdT Polymerization

1. 5'-end labeling of the primer
   1. Mix 30 pmol of the appropriate primer (e.g. P1, List of Materials) with 4 µl of 10x Polynucleotide kinase (PNK) buffer, 3 µl of γ-[³²P]-ATP, 1 µl of PNK, and ddH₂O (for a total reaction volume of 40 µl). Incubate the reaction mixture at 37 °C for 30 min and then heat-deactivate the kinase (10 min at 70 °C).
   2. During the labeling reaction prepare a G10 Sephadex column by plugging a 1 ml pipette tip with silanized glass wool and filling the tip with G10 solution (10% in ddH₂O, autoclaved). Spin down and wash three times with 500 µl ddH₂O and one final wash with 50 µl ddH₂O. Run the reaction mixture through the G10 (to remove the free radioactive label).
   3. Gel purify (PAGE 20%) the radiolabeled primer and recover by application of the crush and soak method (i.e. elution with 500 µl of an aqueous solution containing 1% LiClO₄ and 1 mM NEt₃ (pH 8) at 72 °C for 15 min). Ethanol precipitate and G10 desalt the eluted oligonucleotide.
2. Primer extension reaction
   1. Anneal 1 pmol of radiolabeled primer P1, 10 pmol primer P1, and 10 pmol of template T1 (List of Materials) in 10x reaction buffer (provided by the supplier of the polymerase to be used) by placing the tube in hot (90-95 °C) water and by allowing to gradually cool down to room temperature (over 45 min).
   2. Put the tube on ice and add (in turn) the dNTP cocktail (containing both the modified and the natural triphosphates, 100 µM final concentration) and 1 U of the DNA polymerase (Vent (exo^-), Klenow, Pwo or 9°N_m) and complete with water (for a total reaction volume of 20 µl). Incubate at the optimal working temperature of the enzyme (e.g. 60 °C for 30 min when Vent (exo^-) is being used).
   3. Add 20 µl of stop solution (formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mM), bromophenol blue (0.1%), xylene cyanol (0.1 %)), heat the samples (95 °C, 5 min), cool down (0 °C), and resolve by denaturing polyacrylamide gel electrophoresis. Visualize by phosphorimager.
3. TdT polymerization
   1. Dilute 7 pmol of single-stranded, unlabeled primer P2 (List of Materials), 1 pmol of radiolabeled primer in 1 µl TdT buffer 10x.
   2. Put the tube on ice and add (in turn) the dNTP cocktail (at an adequate concentration comprised between 10 and 200 µM) and the TdT polymerase (4 U). Incubate at 37 °C for 1 hr. Add 10 µl of the stop solution, heat the samples (95 °C, 5 min), cool down (0 °C), and resolve by denaturing polyacrylamide gel electrophoresis (PAGE 15%). Visualize by phosphorimager.

4. PCR with Modified Nucleoside Triphosphates

1. Mix 8 pmol of both the forward and reverse primers with 0.5 pmol of the template to be amplified. Add 10x reaction buffer and the dNTP cocktail (for a 200 µM final concentration) and put the tube on ice. Add 1 U of the DNA polymerase and complete with H₂O to reach a final volume of 20 µl. Transfer the mixture into a PCR vial and carry out 30 PCR cycles.
2. Dilute 6 µl with 6 µl of 2x sucrose loading buffer and resolve by agarose 2% (stained with ethidium bromide) electrophoresis. Visualize by phosphorimager.

Wyniki

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...

Dyskusje

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...

Materiały

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
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.