Name: chemical triphosphorylation of oligonucleotides
Uploaded: 2022-06-02T13:40:00
Duration: 13 min 19 s
Description: Watch this Scientific Journal Video about Chemical Triphosphorylation of Oligonucleotides at JoVE.com

The authors are grateful to Greg Springsteen, Natasha Paul, Charles Olea, Jr., Jonathan Sczepanski, and Katrina Tjhung for useful discussions on best practices for chemical triphosphorylation reactions and to Gerald Joyce for helpful comments. This work was supported by grant MCB 2114588 from the National Science Foundation.

The authors declare that they have no competing financial interests.

The triphosphorylation procedure described here is broadly compatible with oligonucleotide synthesis using standard phosphoramidite chemistry. Nucleoside phosphoramidites should have base-labile protecting groups compatible with rapid deprotection in AMA39, including the standard &#946;-cyanoethyl on the phosphite, and isobutyryl, dimethylformamidyl, acetyl, phenoxyacetyl, or 4-isopropylphenoxyacetyl groups on the exocyclic amines of the nucleobases. Ribose 2&#39;-hydroxyl groups should be protected by silyl protecting groups, either t-butyldimethylsilyl (TBDMS) or tri-iso-propylsilyloxymethyl (TOM)40,41. The base-labile pivaloyloxymethyl (PivOM) group also has been reported to be compatible with chemical triphosphorylation30.
Multiple methods have been described for the chemical triphosphorylation of synthetic oligonucleotides28,29,30,31,32,33,34,35. We have found triphosphorylation using the Ludwig-Eckstein reagent37 to be one of the most accessible, requiring no specialized synthesis of reagents and no specialized equipment. Oligonucleotide 5&#8242;-triphosphates prepared by this method have been used routinely as substrates for RNA ligase ribozymes, including the use of enzymatically inaccessible L-RNA oligonucleotide triphosphates to achieve template-dependent synthesis and replication of this &#34;mirror-image&#34; nucleic acid14,16,17,18. The method is also suitable for the preparation of small 5&#8242;-triphosphorylated stem-loop RNAs that are potent activators of the innate immune response in vertebrates6,7.
The Ludwig-Eckstein reagent, salicyl phosphorochloridite37, is highly reactive to water, and effectively scavenges any water introduced when dissolving the reagent prior to loading onto the oligonucleotide column. After this point, however, the 5&#8242;-phosphitylated oligonucleotide will preferentially react with any water introduced into the system over pyrophosphate, forming a 5&#8242;-H-phosphonate side product after workup28,37,38. Careful preparation of triphosphorylation reagents and the triphosphorylation reaction chamber ensure this side product is not formed. For solvent drying, type 4 &#197; molecular sieves are sold prepackaged in Teflon bags compatible with most organic solvents under various brand names by most oligonucleotide synthesis reagent companies. Additional precautions, such as performing triphosphorylation in a glove box under an anhydrous atmosphere, are generally not necessary.
Reaction of the 5&#8242;-phosphitylated oligonucleotide with TBAP forms a cyclic 5&#8242;-trimetaphosphite intermediate, which is then oxidized to the cyclic 5&#8242;-trimetaphosphate using oligonucleotide synthesis oxidizer solution (iodine in water/pyridine/THF). It should be noted that commercial oxidizer solutions use varying amounts of iodine, and it is essential to use the high 0.1 M iodine concentration to ensure complete oxidation to the triphosphate. The cyclic product is hydrolyzed to the final linear 5&#8242;-triphosphate in the same solution37, and alternative, anhydrous oxidizer solutions must be used if linearization with nucleophiles other than water is desired (see below for applications)33. Any residual cyclic trimetaphosphate, however, will be linearized during subsequent alkaline deprotection of the oligonucleotide. Hydrolysis of the cyclic 5&#8242;-trimetaphosphate yields only the linear, rather than branched triphosphate37,46.
Oligonucleotide deprotection typically does not need to be modified to accommodate 5&#8242;-triphosphorylation, but a few precautions should be taken. The triphosphate is relatively stable to brief exposure to alkaline conditions, but care should be taken not to expose the triphosphate to AMA longer than necessary. Protecting groups that require more prolonged treatment in ammonia or AMA for more than 10 min at 65 &#176;C should be avoided. More gentle treatments, such as 2 h in ammonia at room temperature are acceptable when compatible with other phosphoramidite protecting groups. A common, fast deprotection method for silyl-protected synthetic RNA oligonucleotides uses triethylamine trihydrofluoride and high temperature47; however, this should be avoided when preparing RNA 5&#8242;-triphosphates as the prolonged acidic conditions are found to accelerate triphosphate hydrolysis31,32.
Preparative PAGE has proven to be the simplest and most reliable method for postdeprotection purification of 5&#8242;-triphosphorylated oligonucleotides (Figure 3 and Figure 4). However, preparative reverse-phase HPLC can also be used to purify triphosphorylated products. The presence of 5&#8242;-diphosphate and, to a lesser extent, 5&#8242;-monophosphate products is routinely observed when verifying triphosphorylation by mass spectrometry. We have observed 5&#8242;-triphosphate fragmentation during mass spectrometry from highly pure material prepared by chemical synthesis or transcription, particularly if the instrument is not optimized for analysis of oligonucleotides. Nevertheless, RP-LC analysis often shows 10%-20% of the 5&#8242;-diphosphate side product is present in longer 5&#8242;-triphosphorylated oligonucleotides (Figure 4). Commercial preparations of tributylammonium pyrophosphate can be contaminated with as much as 20% monophosphate, which will yield 5&#8242;-diphosphate as a side product during triphosphorylation30,31. Careful preparation of this reagent in-house can yield much more pure TBAP stocks31. However, we have found oligonucleotides triphosphorylated using commercial sources of TBAP still show comparable or greater reactivity when used as substrates in enzymatic reactions (Figure 5B), compared to material prepared by in vitro transcription.
One notable further use of oligonucleotide triphosphorylation with the Ludwig-Eckstein reagent takes advantage of the cyclic trimetaphosphite intermediate33. If the subsequent oxidation step is conducted with 1 M t-butyl peroxide in hexanes, which is often used for oligonucleotide oxidation under anhydrous conditions, oxidation of the phosphite occurs without ring opening hydrolysis, yielding the cyclic trimetaphosphate. This intermediate can then be reacted with primary amine or alcohol nucleophiles to yield 5&#8242;-triphosphates with modifications at the &#947;-phosphate. These modifications include the addition of a lipophilic tag linked by a phosphoramidate bond, which facilitates rapid triphosphate-specific purification by RP-LC, followed by acidic hydrolysis of the tag from the triphosphate33. Fluorescent modifications at the &#947;-phosphate position can also be introduced for use as real-time fluorescent reporters for ribozyme-catalyzed ligation reactions15,33.

The 5&#8242;-triphosphorylated form of RNA is ubiquitous in biology as it is generated by RNA transcription in all domains of life and by RNA replication during the life cycle of many RNA viruses. These triphosphates serve as the substrate for the formation of 7-methylguanylate-capped mRNA in eukaryotes and, therefore, play an essential role in protein expression1. In contrast, the triphosphate is retained in bacteria and viruses; thus, RNA 5&#8242;-triphosphates are recognized by innate immunity response regulators in eukaryotes2,3,4,5,6,7. Outside biology, a host of RNA ligase ribozymes have been evolved to use the 5&#8242;-triphosphate in vitro8&#160;and modified for use in diagnostic assays9,10,11,12,13,14,15. One such ribozyme can be used for template-dependent synthesis of L-RNA, the nonbiological &#34;mirror-image&#34; enantiomer of natural D-RNA, from small L-RNA oligonucleotide 5&#8242;-triphosphates16,17,18. The routine preparation of triphosphorylated oligonucleotides of varying sequence and backbone composition is essential to investigating these systems.
The most common and accessible method for preparing RNA 5&#8242;-triphosphates in the laboratory is by in vitro transcription. However, RNA produced by this method is restricted in sequence and size by promoter and substrate requirements of the RNA polymerase enzyme. T7 RNA polymerase and specialized derivatives are the most common polymerases used for this purpose19,20,21,22. In vitro transcribed RNA prepared with these enzymes must be initiated with a 5&#8242;-terminal purine and is strongly biased toward purines in the first 10 nucleotides23,24. Moreover, enzymatic incorporation of base- or backbone-modified nucleotides is at best inefficient and more often impossible with natural polymerases, limiting the opportunity to produce oligonucleotide 5&#8242;-triphosphates composed of anything but natural D-RNA. Another limiting factor is that RNA generated by in vitro transcription can contain substantial 5&#8242;- and 3&#8242;- heterogeneity and is produced as extremely heterogeneous products when shorter than 20 nt23,24,25,26,27.
In contrast, chemical triphosphorylation of oligonucleotides prepared by solid-phase phosphoramidite synthesis28,29,30,31,32,33,34,35 can be used to prepare oligonucleotide 5&#8242;-triphosphates 3-50 nt long, of any sequence. Additionally, a vast array of nucleic acid modifications accessible to phosphoramidite synthesis can be added to oligonucleotides prior to 5&#8242;-triphosphorylation14,15,16,17,18,29,36. Many of these methods use the phosphitylation reagent salicyl phosphorochloridite, which was developed by Ludwig and Eckstein for the solution-phase triphosphorylation of mononucleosides37. Triphosphorylation of oligonucleotides with this reagent is achieved on the solid phase by phosphitylation of the oligonucleotide 5&#8242;-hydroxyl, conversion to the triphosphate by reaction with pyrophosphate and oxidation, followed by standard procedures for cleavage of the oligonucleotide from the solid support, deprotection, and purification (Figure 1)28.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63877/63877fig01v2.jpg" /> 
Figure 1: Scheme for triphosphorylation of synthetic oligonucleotides. In the first step, the oligonucleotide 5&#697;-hydroxyl is phosphitylated with SalPCl. In the next step, the 5&#697;-salicyl phosphite is reacted with TBAP to form the cyclic metaphosphite, then in the third step oxidized to generate the cyclic 5&#697;-trimetaphosphate in DNA/RNA synthesizer oxidation solution (0.1 M Iodine/pyridine/H2O/THF), which is rapidly hydrolyzed to yield the linear 5&#697;-triphosphate in the same solution28,33,37. Subsequent alkaline cleavage from the solid CPG support and deprotection of the oligonucleotide in aqueous MeNH2/ammonia will hydrolyze any residual cyclic trimetaphosphate to the linear form. Abbreviations: SalPCl = salicyl phosphorochloridite; TBAP = tributylammonium pyrophosphate; THF = tetrahydrofuran; CPG = controlled pore glass; MeNH2 = methylamine. <a href="https://www.jove.com/files/ftp_upload/63877/63877fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although early published reports using this method often suffered from poor yields and undesired side products28,37,38, careful maintenance of anhydrous conditions is all that is necessary for routinely obtaining high yields. This can be achieved by careful preparation of reagents and the use of a simple reaction device assembled from standard plastic components. Here, we demonstrate the appropriate steps for chemical triphosphorylation of oligonucleotides, including preparation of reagents, assembly of the reaction chamber, the triphosphorylation reaction, and subsequent deprotection and purification of the triphosphorylated oligonucleotides. Also included is the representative use of 5&#8242;-triphosphorylated oligonucleotides as substrates for ligase ribozymes for the synthesis of larger nucleic acid products with natural D-RNA and abiotic L-RNA backbones.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m polyethersulfone syringe filter</td>
			<td>MilliporeSigma</td>
			<td>SLMP025SS</td>
			<td>Syringe filter for removing crushed polyacrylamide gel particles (Section 5)</td>
		</tr>
		<tr>
			<td>0.22 &#181;m PTFE syringe filter</td>
			<td>MilliporeSigma</td>
			<td>SLLG013SL</td>
			<td>Syringe filter for removing CPG resin (Section 5)</td>
		</tr>
		<tr>
			<td>1 mL plastic syringes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-823-434 (BD 309659)</td>
			<td>Components of triphosphorylation apparatus (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>1,4-Dioxane, anhydrous</td>
			<td>MilliporeSigma</td>
			<td>296309</td>
			<td>Triphosphorylation solvent (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, Salicyl Phosphorochloridite (SalPCl)</td>
			<td>MilliporeSigma</td>
			<td>324124</td>
			<td>Triphosphorylation reagent (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>30 mL glass bottles</td>
			<td>MilliporeSigma</td>
			<td>23232</td>
			<td>Bottles for preparing triphosphorylation solvents and TBAP solution (section 2)</td>
		</tr>
		<tr>
			<td>3-way Stopcock, polycarbonate/polypropylene</td>
			<td>Bio-Rad Laboratories</td>
			<td>7328103</td>
			<td>Component of triphosphorylation apparatus (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>40% acrylamide/bis-acrylamide solution, 19:1</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610144</td>
			<td>For PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Acetonitrile, anhydrous, 100 mL</td>
			<td>Glen Research</td>
			<td>40-4050-50</td>
			<td>Triphosphorylation solvent (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>Ammonia-neutralizing Trap</td>
			<td>ThermoFisher Scientific</td>
			<td>ANT100 and ANS121</td>
			<td>For use with Speedvac DNA130 (section 5)</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610700</td>
			<td>For PAGE, catalyst for acrylamide polymerization (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Aqueous ammonia, 28%</td>
			<td>MilliporeSigma</td>
			<td>338818</td>
			<td>For preparing AMA deprotection reagent (section 5)</td>
		</tr>
		<tr>
			<td>Aqueous methylamine, 40%</td>
			<td>TCI America</td>
			<td>TCI-M0137</td>
			<td>For preparing AMA deprotection reagent (section 5)</td>
		</tr>
		<tr>
			<td>Automated DNA/RNA oligonucleotide synthesizer</td>
			<td>PerSeptive Biosystems</td>
			<td>Expedite 8909 DNA/RNA Synthesizer</td>
			<td>any column-based synthesizer is acceptable (section 1)</td>
		</tr>
		<tr>
			<td>Bead-capture magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12320D</td>
			<td>For streptavidin bead capture (section 7)</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610404</td>
			<td>For PAGE urea loading buffer (section 5)</td>
		</tr>
		<tr>
			<td>Deep vacuum oil pump</td>
			<td>ThermoFisher Scientific</td>
			<td>VLP200-115</td>
			<td>For use with lyophilizer (section 5)</td>
		</tr>
		<tr>
			<td>Drierite dessicant, 10-20 mesh</td>
			<td>MilliporeSigma</td>
			<td>737828</td>
			<td>Desiccant for storing triphosphorylation chemicals and equipment (sections 1&#8211;2)</td>
		</tr>
		<tr>
			<td>D-RNA 27.3t cross-chiral polymerase</td>
			<td>prepared in house18</td>
			<td>5&#8242;-GGUGGUGGAC GUGAUCAUUA CGGAUCACUA ACUCGUCAGU GCAUUGAGAA GGAGAAUAAA AUGCACAUAG GUCGAAAGAC CUUAUACAAG AACUGUAUCA CCGGAGGGCG AGCACCACC-3&#8242;</td>
			<td>For cross-chiral ribozyme reactions (section 7)</td>
		</tr>
		<tr>
			<td>D-RNA CPG solid supports, 1,000&#197;, prepackaged 1 &#181;mole synthesis columns</td>
			<td>Glen Research</td>
			<td>20-3404-41E, 20-3415-41E, 20-3424-41E, 20-3430-41E</td>
			<td>representative, for D-RNA oligonucleotide synthesis (section 1)</td>
		</tr>
		<tr>
			<td>D-RNA TOM-protected phosphoramidites</td>
			<td>ChemGenes</td>
			<td>ANP-3201, 3202, 3203, 3205</td>
			<td>representative, for D-RNA oligonucleotide synthesis (section 1)</td>
		</tr>
		<tr>
			<td>Empty Expedite Synthesis Columns, 1&#181;m</td>
			<td>Glen Research</td>
			<td>20-0021-01</td>
			<td>Synthesis columns for use with Expedite DNA/RNA synthesizer (section 1)</td>
		</tr>
		<tr>
			<td>EPPS, N-(2-Hydroxyethyl)piperazine-N&#8242;-(3-propanesulfonic acid), solid</td>
			<td>MilliporeSigma</td>
			<td>E1894</td>
			<td>Ribozyme reaction buffer component (section 6)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA), solid</td>
			<td>MilliporeSigma</td>
			<td>EDS</td>
			<td>Divalent metal ion chelator for use in various buffers (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Filters for Expedite synthesis columns</td>
			<td>Glen Research</td>
			<td>20-0021-0F</td>
			<td>Expedite-style synthesis column filters, for use with empty synthesis columns (section 1)</td>
		</tr>
		<tr>
			<td>Fluorescent/phosphorescent gel scanner</td>
			<td>Cytiva</td>
			<td>Amersham Typhoon RGB, 29187193</td>
			<td>For visualizing analytical PAGE (sections 6&#8211;7)</td>
		</tr>
		<tr>
			<td>Formamide, deionized</td>
			<td>VWR Life Science</td>
			<td>97062</td>
			<td>For PAGE formamide gel loading buffer (sections 6&#8211;7)</td>
		</tr>
		<tr>
			<td>Gel image quantitation software</td>
			<td>Cytiva</td>
			<td>ImageQuant TL</td>
			<td>For quantifying scanned gel images (section 6)</td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>MilliporeSigma</td>
			<td>CLS3121150</td>
			<td>Triphosphorylation solvent storage (section 2)</td>
		</tr>
		<tr>
			<td>L-RNA CPG solid supports, 1,000&#197;, bulk</td>
			<td>ChemGenes</td>
			<td>N-4691-10, N-4692-10, N-4693-10, N-4694-10</td>
			<td>L-RNA oligonucleotide synthesis (section 1)</td>
		</tr>
		<tr>
			<td>L-RNA hammerhead template</td>
			<td>prepared in house18</td>
			<td>5&#8242;-GCGCCUCAUC AGUCGAGCC-3&#8242;</td>
			<td>For cross-chiral ribozyme reactions (section 7)</td>
		</tr>
		<tr>
			<td>L-RNA primer</td>
			<td>prepared in house18</td>
			<td>5&#8242;-fluorescein-GGCUCGA-3&#8242;</td>
			<td>For cross-chiral ribozyme reactions (section 7)</td>
		</tr>
		<tr>
			<td>L-RNA TOM-protected phosphoramidites</td>
			<td>ChemGenes</td>
			<td>OP ANP-5201, 5202, 5203, 5205</td>
			<td>L-RNA oligonucleotide synthesis (section 1)</td>
		</tr>
		<tr>
			<td>Lyophilizer/Freeze Dryer</td>
			<td>VirTis</td>
			<td>Benchtop K</td>
			<td>For concentrating oligonucleotides (section 5)</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate, solid</td>
			<td>MilliporeSigma</td>
			<td>M2670</td>
			<td>For ribozyme reactions (sections 6&#8211;7)</td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, anhydrous</td>
			<td>MilliporeSigma</td>
			<td>227056</td>
			<td>Triphosphorylation solvent (section 2)</td>
		</tr>
		<tr>
			<td>NAP-25 Desalting column (Sephadex G-25 resin)</td>
			<td>ThermoFisher Scientific</td>
			<td>45000150</td>
			<td>Disposable gravity-flow size exclusion chromatography columns containing Sephadex G-25 resin (section 5)</td>
		</tr>
		<tr>
			<td>Non-coring stainless steel needle, 20 G</td>
			<td>ThermoFisher Scientific</td>
			<td>14-815-410</td>
			<td>Needles for piercing rubber septa (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>Oligonucleotide extinction coefficient calculator</td>
			<td>Integrated DNA Technologies</td>
			<td>OligoAnalyzer Tool</td>
			<td>Nearest-Neighbor Model Short Oligonucleotide 260nm extinction coefficient calculator (section 5)</td>
		</tr>
		<tr>
			<td>Oxidizer solution, 0.1 M Iodine in THF/pyridine/water</td>
			<td>ChemGenes</td>
			<td>RN-1456</td>
			<td>Triphosphorylation reagent (section 4)</td>
		</tr>
		<tr>
			<td>PAGE plates</td>
			<td>Timberrock/CBS</td>
			<td>NGP-250-BO and NO</td>
			<td>For PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>PAGE power supply</td>
			<td>Bio-Rad Laboratories</td>
			<td>PowerPac HV 1645056</td>
			<td>For PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>PAGE spacers and combs (analytical)</td>
			<td>Timberrock/CBS</td>
			<td>VGS-0725 and VGC-0714</td>
			<td>For PAGE (sections 6&#8211;7)</td>
		</tr>
		<tr>
			<td>PAGE spacers and combs (preparative)</td>
			<td>Timberrock/CBS</td>
			<td>VGS-3025R and VGC-3001</td>
			<td>For PAGE (section 5)</td>
		</tr>
		<tr>
			<td>PAGE stand</td>
			<td>Timberrock/CBS</td>
			<td>ASG-250</td>
			<td>For PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>ThermoFisher Scientific</td>
			<td>13-374-12 (Bemis PM999)</td>
			<td>Wax sealing film for triphosphorylation apparatus (sections 2&#8211;4)</td>
		</tr>
		<tr>
			<td>PCR thermocycler</td>
			<td>Bio-Rad Laboratories</td>
			<td>C1000 Touch Thermalcycler</td>
			<td>For cross-chiral ribozyme reactions (section 7)</td>
		</tr>
		<tr>
			<td>PD 10 Desalting column (Sephadex G-10 resin)</td>
			<td>MilliporeSigma</td>
			<td>GE17-0010-01</td>
			<td>Disposable gravity-flow size exclusion chromatography columns containing Sephadex G-10 resin, for oligonucleotides &#60; 15 nt (section 5)</td>
		</tr>
		<tr>
			<td>Phosphor screens</td>
			<td>Cytiva</td>
			<td>28956480</td>
			<td>For visualizing 32P-labeled RNA (section 6)</td>
		</tr>
		<tr>
			<td>Phosphoramidite synthesis reagents</td>
			<td>Glen Research</td>
			<td>30-3142-52, 40-4050-53, 40-4012-52, 40-4122-52, 40-4132-52, 40-4060-62</td>
			<td>representative, for standard RNA/DNA synthesis (section 1)</td>
		</tr>
		<tr>
			<td>Polypropylene screw-cap sealable tube</td>
			<td>MilliporeSigma</td>
			<td>BR780752</td>
			<td>1.5 mL microcentrifuge tubes with screw-cap and silicone O-ring, for safe AMA deprotection (section 5)</td>
		</tr>
		<tr>
			<td>Pyridine, anhydrous</td>
			<td>MilliporeSigma</td>
			<td>270970</td>
			<td>Triphosphorylation solvent (section 2)</td>
		</tr>
		<tr>
			<td>Reverse-phase liquid chromatography/electrospray ionization mass spectrometry (RP-LC/ESI-MS)</td>
			<td>Novatia</td>
			<td>n/a</td>
			<td>Commercial service for LC/MS specializing in oligonucleotides (section 5)</td>
		</tr>
		<tr>
			<td>Rubber Septa (ID x OD 7.9 mm x 14 mm), white</td>
			<td>MilliporeSigma</td>
			<td>Z564702</td>
			<td>Septa for preparing triphosphorylation solvents and TBAP (section 2)</td>
		</tr>
		<tr>
			<td>Self-replicator ribozyme E</td>
			<td>prepared in house14</td>
			<td>5&#8242;-GGAAGUUGUG CUCGAUUGUU ACGUAAGUAA CAGUUUGAAU GGUUGAAGUA UGAGACCGCA ACUUA-3&#8242;</td>
			<td>For self-replicator ribozyme reactions (section 6)</td>
		</tr>
		<tr>
			<td>Self-replicator substrate A</td>
			<td>prepared in house14</td>
			<td>5&#8242;-32P-GGAAGUUGUG CUCGAUUGUU ACGUAAGUAA CAGUUUGAAU GGUUGAAGUA U-3&#8242;-OH</td>
			<td>For self-replicator ribozyme reactions (section 6)</td>
		</tr>
		<tr>
			<td>Self-replicator substrate B, transcribed</td>
			<td>prepared in house14</td>
			<td>5&#8242;-pppGAGACCGCAA CUUA-3&#8242;</td>
			<td>For self-replicator ribozyme reactions (section 6)</td>
		</tr>
		<tr>
			<td>Small Drying Traps, 4 &#197; molecular sieves</td>
			<td>ChemGenes</td>
			<td>DMT-1975</td>
			<td>Drying traps for DNA/RNA synthesizer phosphoramidites and triphosphorylation reagents (sections 1&#8211;2)</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl), solid</td>
			<td>MilliporeSigma</td>
			<td>S7653</td>
			<td>Salt for use in various buffers (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH), solid</td>
			<td>MilliporeSigma</td>
			<td>S8045</td>
			<td>Salt for use in various buffers (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Statistical data-fitting software</td>
			<td>GraphPad</td>
			<td>Prism</td>
			<td>For fitting data from analytical PAGE to kinetic models (section 6)</td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic beads</td>
			<td>ThermoFisher Scientific</td>
			<td>65002</td>
			<td>For capturing biotin-labeled RNA in cross-chiral ribozyme reactions (section 7)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>MilliporeSigma</td>
			<td>84097</td>
			<td>For PAGE urea loading buffer (section 5)</td>
		</tr>
		<tr>
			<td>TBE running buffer, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ62788K3</td>
			<td>For PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Tetrabutylammonium Fluoride, 1.0 M solution in Tetrahydrofuran</td>
			<td>Aldrich</td>
			<td>216143</td>
			<td>For removing 2&#8242;-silyl protecting groups (section 5)</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610801</td>
			<td>For polymerizing acrylamide for PAGE (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>Tributylamine</td>
			<td>MilliporeSigma</td>
			<td>90781</td>
			<td>Triphosphorylation reagent (section 2)</td>
		</tr>
		<tr>
			<td>Tributylammonium pyrophosphate (TBAP)</td>
			<td>MilliporeSigma</td>
			<td>P8533</td>
			<td>Triphosphorylation reagent (section 2)</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>MilliporeSigma</td>
			<td>T6666</td>
			<td>Buffering agent for use in various buffers (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>TWEEN20 polysorbate detergent</td>
			<td>MilliporeSigma</td>
			<td>P7949</td>
			<td>Neutral detergent for use with magnetic beads (Section 7)</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>MilliporeSigma</td>
			<td>U5378</td>
			<td>For PAGE and gel loading buffer (sections 5&#8211;7)</td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>NanoDrop 2000, ND2000</td>
			<td>For measuring oligonucleotide concentrations (section 5)</td>
		</tr>
		<tr>
			<td>Vacuum centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>Savant Speedvac DNA130-115 Vacuum Concentrator</td>
			<td>For removing AMA and THF (section 5)</td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610423</td>
			<td>For PAGE urea loading buffer (section 5)</td>
		</tr>
	</tbody>
</table>

chemical triphosphorylation of oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotides should be synthesized using standard protocols appropriate to the phosphoramidites and automated DNA/RNA synthesizer, leaving the product oligonucleotide uncleaved from the solid support in the original plastic synthesis column, with the 5&#697;-terminal dimethoxytrityl group removed to yield the free 5&#697;-hydroxyl (section 1). All oligonucleotides used in this demonstration were prepared using 1,000 &#197; controlled pore glass (CPG) resin as the solid support and conducted at the 0.2 or 1 &#181;mole scale. Representative examples of synthesizer columns, resins, reagents, and phosphoramidites are provided in the Table of Materials. For larger-scale reactions, volumes and times used in subsequent steps may need to be adjusted.
The triphosphorylation reaction is conducted on-column in a custom-built reaction chamber (Figure 2, section 3) using standard, commercially available components listed in the Table of Materials and follows the scheme illustrated in Figure 1 (section 4)28. It is essential that conditions be kept strictly anhydrous during triphosphorylation, and that all solvents and reagents be prepared over molecular sieves in advance and allowed to fully dry before use (section 2). Triphosphorylation typically takes 2 h to occur, and afterward, the washed and dried column can be treated according to standard oligonucleotide deprotection and purification procedures (section 5).
After deprotection, oligonucleotide triphosphates are purified by denaturing polyacrylamide gel electrophoresis (PAGE), showing a single major product band by UV back-shadowing that can be excised and eluted from the gel. The 5&#8242;-triphosphate product is readily separated from reaction side products for short oligonucleotides, as shown for DNA trinucleotide 5&#697;-triphosphates, pppAAA and pppCCC, and L-RNA trinucleotide 5&#697;-triphosphate pppGAA in Figure 3A,B. Both the 5&#8242;-hydroxyl and 5&#8242;-triphosphate products for AAA and CCC DNA trimers were excised and identified by mass spectrometry and correspondingly labeled in Figure 3A. Additional bands, as visible for the AAA DNA trimer, generally do not contain enough material to recover and identify. The presence of these bands, however, correlates with additional product masses in the unpurified reaction products (Figure 3C), typically representing 5&#8242;-diphosphate, monophosphate, and H-phosphonate side products, as discussed below.
After PAGE purification, larger oligonucleotides can be eluted using the crush and soak method42 and subsequent ethanol precipitation. However, oligonucleotides less than 15 nt cannot be ethanol precipitated efficiently and, thus, require a modified procedure for gel elution (step 5.11.3). The disposable size exclusion column listed in the Table of Materials is rated only for use with oligonucleotides longer than 10 nt. However, we have found that oligonucleotides as short as trimers can be effectively desalted using the manufacturer&#39;s recommended protocol. Nevertheless, it is recommended when desalting short oligonucleotides (as in steps 5.6 and 5.11.3) that the column eluate be collected in fractions, and product fractions be identified by absorbance at 260 nm using a UV-Vis spectrophotometer. A size exclusion column optimized for shorter oligonucleotides is provided in the Table of Materials as an alternative choice. The final yield from 1 &#181;mole scale oligonucleotide synthesis after purification is 50-300 nmol.
Triphosphorylation can be confirmed by mass spectrometry, where the triphosphorylated product has a mass +239.94 Da greater than the 5&#8242;-hydroxyl oligonucleotide, although the presence of materials corresponding to the 5&#8242;-di- and monophosphate (+159.96 and +79.98 Da, respectively) are often observed. A 5&#8242;-H-phosphonate side product with a mass +63.98 Da from the 5&#8242;-OH mass may also be observed, and high levels of this product indicate conditions during triphosphorylation were not sufficiently anhydrous. Prior to purification, deprotected oligonucleotides will typically show all these products (Figure 3C), while purified material will show a peak corresponding to the 5&#8242;-triphosphate product along with 5&#8242;-di- and monophosphates (Figure 3D,E).
Mass spectrometry alone will typically not give a rigorous measure of 5&#8242;-triphosphate purity due to differential rates of ionization and fragmentation of the triphosphate during ionization. To measure final product purity, reverse-phase liquid chromatography and tandem ESI-MS (RP-LC/ESI-MS) are recommended, particularly for longer oligonucleotides. Analysis of D-RNA 5&#697;-triphosphates pppACGAGG and pppGAGACCGCAACUUA by RP-LC/ESI-MS (Figure 4A,B, respectively) show typical final product purity, containing 20% 5&#697;-diphosphate as these two species are difficult to separate when present on longer oligonucleotides.
Synthetic 5&#8242;-triphosphate oligonucleotides typically function as well or better than materials prepared enzymatically in biochemical studies. In section 6, as an example, 5&#8242;-triphosphate 14 nt RNA substrates prepared either synthetically or by in vitro transcription were compared in an RNA-catalyzed self-replication reaction14,15,43,44,45. Ribozyme E catalyzes the joining of substrates A and B to yield a new copy of E in an autocatalytic reaction capable of exponential growth (Figure 5A). E and 32P-labeled A components were prepared by in vitro transcription, and triphosphorylated substrate B was prepared either synthetically, as described above, or by in vitro transcription14. Self-replication reaction progress was monitored by taking periodic samples that were analyzed by denaturing PAGE and quantified via a fluorescent/phosphorescent gel scanner. The resulting data, fit to a logistic growth function, revealed that either transcribed or synthetic B substrate supports exponential growth, but synthetic B gives a slightly greater amount of product (Figure 5B). This result may reflect compositional heterogeneity at the 5&#8242;-end of RNA prepared by in vitro transcription23,24.
Chemical triphosphorylation also enables the synthesis of oligonucleotide triphosphates that cannot be prepared biologically, either in vitro or in cells. In section 7, nonbiological oligonucleotide triphosphates composed of L-RNA, the enantiomer of natural D-RNA, prepared as in sections 1-5, were used as substrates for the D-RNA &#34;cross-chiral&#34; polymerase ribozyme 27.3t (Figure 6A), which catalyzes the template-directed polymerization of a longer L-RNA product from short L-RNA oligonucleotide 5&#8242;-triphosphates in a sequence-general manner. As an example, the ribozyme can synthesize an L-RNA version of the hammerhead self-cleavage motif (Figure 6B)18. Purified L-RNA trinucleotide triphosphates were combined with a fluorescein-labeled L-RNA primer and L-RNA template (Figure 6C) and reacted with the cross-chiral ligase. Samples over the course of the reaction were analyzed by PAGE and imaged using a fluorescent/phosphorescent gel scanner to demonstrate synthesis of an L-RNA version of the hammerhead ribozyme encoded by the template (Figure 6D).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63877/63877fig03.jpg" /> 
Figure 3: Purification of trinucleotide 5&#697;-triphosphates. (A) PAGE analysis (visualized by UV-back-shadowing) of triphosphorylation of DNA trinucleotides tri-deoxyadenosine (AAA, blue) and tri-deoxycytidine (CCC, red), intentionally overloaded to visualize minor side-products. Both the 5&#697;-triphosphate product (ppp) and 5&#697;-hydroxyl (OH) starting material were excised and identified by MALDI-MS. (B) Preparative PAGE of triphosphorylation of L-RNA trinucleotide GAA, with major product band excised and identified as the 5&#697;-triphosphate (ppp) by ESI-MS. (C) MALDI-MS of crude reaction products after deprotection and (D) purified products from (A). 5&#697;-triphosphate (ppp; pppAAA expected 1,119 Da, observed 1,118 Da; pppCCC expected 1,047 Da, observed 1,046); 5&#697;-diphosphate (pp), 5&#697;-monophosphate (p), 5&#697;-hydroxyl (OH), and 5&#697;-H-phosphonate (Hp) are labeled. (E) Deconvoluted mass spectrum from direct injection ESI-MS of isolated 5&#697;-triphosphate product from (B), with identified peaks labeled (expected 1,181.6 Da, observed 1,181.0 Da). 5&#697;-diphosphate (pp) products are also observed, as are sodium ion peaks for both the tri- and di-phosphate products (+22 Da). Common contaminant peaks are labeled with an asterisk. For ease of comparison, mass spectra were normalized to the highest intensity measured in each spectrum and are reported as a percentage relative to that value. Abbreviations: PAGE = polyacrylamide gel electrophoresis; MALDI-MS = matrix-assisted laser desorption/ionization; ESI-MS = electrospray ionization mass spectrometry. <a href="https://www.jove.com/files/ftp_upload/63877/63877fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63877/63877fig04.jpg" /> 
Figure 4: Analytical RP-LC of 6 nt and 14 nt D-RNA oligonucleotide triphosphates. (A) 5&#697;-pppACGAGG-3&#697; and (B) 5&#697;-pppGAGACCGCAACUUA-3&#697;. Tandem ESI-MS identified the major peak of both (~70%) as the 5&#697;-triphosphate (ppp), with lesser amounts of the 5&#697;-diphosphate (pp). Abbreviations: RP-LC = reverse-phase liquid chromatography; nt = nucleotides; ESI-MS = electrospray ionization mass spectrometry. <a href="https://www.jove.com/files/ftp_upload/63877/63877fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63877/63877fig05.jpg" /> 
Figure 5: Comparison of oligonucleotide 5&#697;-triphosphate substrates prepared by chemical synthesis or in vitro transcription. (A) The self-replicating ribozyme E ligates RNA A and 5&#8242;-triphosphorylated RNA B. (B) Comparison of self-replication reactions using 10 &#181;M A and 10 &#181;M B, either synthetic (open circles) or in vitro transcribed (filled circles). (B) Data were fit to the logistic growth equation: [E] = a / (1 + be-ct), where a is the final yield, b is the degree of sigmoidicity, and c is the exponential growth rate. Growth rates for the two reactions were identical, at 1.14 h-1, while the final extent was 10% higher for reactions with synthetic B. <a href="https://www.jove.com/files/ftp_upload/63877/63877fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63877/63877fig06.jpg" /> 
Figure 6: Cross-chiral L-RNA polymerization with a ribozyme. (A) The D-RNA 27.3t polymerase ribozyme, which catalyzes template-dependent ligation of L-RNA. (B) The L-RNA product synthesized by 27.3t forms part of a hammerhead endonuclease motif. (C) L-RNA polymerization catalyzed by 27.3t using a biotinylated L-RNA template (brown), an end-labeled L-RNA primer (magenta), and four L-RNA trinucleotide triphosphates (cyan), prepared synthetically. (D) PAGE analysis of extension products of (B) at 4 h and 24 h, showing each trinucleotide incorporation up to full-length product (black dot). Unreacted L-RNA primer is included as a reference marker. Abbreviations: PAGE = polyacrylamide gel electrophoresis; M = reference marker. <a href="https://www.jove.com/files/ftp_upload/63877/63877fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Chemical Triphosphorylation of Oligonucleotides at JoVE.com

Chemical Triphosphorylation of Oligonucleotides

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

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