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><tbody><tr><td>0.22 &amp;micro;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 &amp;micro;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&amp;ndash;4)</td></tr><tr><td>1,4-Dioxane, anhydrous</td><td>MilliporeSigma</td><td>296309</td><td>Triphosphorylation solvent (sections 2&amp;ndash;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&amp;ndash;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&amp;ndash;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&amp;ndash;7)</td></tr><tr><td>Acetonitrile, anhydrous, 100 mL</td><td>Glen Research</td><td>40-4050-50</td><td>Triphosphorylation solvent (sections 2&amp;ndash;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&amp;ndash;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&amp;ndash;2)</td></tr><tr><td>D-RNA 27.3t cross-chiral polymerase</td><td>prepared in house&lt;sup&gt;18&lt;/sup&gt;</td><td>5&amp;prime;-GGUGGUGGAC GUGAUCAUUA CGGAUCACUA ACUCGUCAGU GCAUUGAGAA GGAGAAUAAA AUGCACAUAG GUCGAAAGAC CUUAUACAAG AACUGUAUCA CCGGAGGGCG AGCACCACC-3&amp;prime;</td><td>For cross-chiral ribozyme reactions (section 7)</td></tr><tr><td>D-RNA CPG solid supports, 1,000&amp;Aring;, prepackaged 1 &amp;micro;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&amp;micro;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&amp;prime;-(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&amp;ndash;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&amp;ndash;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&amp;ndash;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&amp;Aring;, 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 house&lt;sup&gt;18&lt;/sup&gt;</td><td>5&amp;prime;-GCGCCUCAUC AGUCGAGCC-3&amp;prime;</td><td>For cross-chiral ribozyme reactions (section 7)</td></tr><tr><td>L-RNA primer</td><td>prepared in house&lt;sup&gt;18&lt;/sup&gt;</td><td>5&amp;prime;-fluorescein-GGCUCGA-3&amp;prime;</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&amp;ndash;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&amp;ndash;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&amp;ndash;7)</td></tr><tr><td>PAGE power supply</td><td>Bio-Rad Laboratories</td><td>PowerPac HV 1645056</td><td>For PAGE (sections 5&amp;ndash;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&amp;ndash;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&amp;ndash;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&amp;ndash;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 &amp;lt; 15 nt (section 5)</td></tr><tr><td>Phosphor screens</td><td>Cytiva</td><td>28956480</td><td>For visualizing &lt;sup&gt;32&lt;/sup&gt;P-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 house&lt;sup&gt;14&lt;/sup&gt;</td><td>5&amp;prime;-GGAAGUUGUG CUCGAUUGUU ACGUAAGUAA CAGUUUGAAU GGUUGAAGUA UGAGACCGCA ACUUA-3&amp;prime;</td><td>For self-replicator ribozyme reactions (section 6)</td></tr><tr><td>Self-replicator substrate A</td><td>prepared in house&lt;sup&gt;14&lt;/sup&gt;</td><td>5&amp;prime;-&lt;sup&gt;32&lt;/sup&gt;P-GGAAGUUGUG CUCGAUUGUU ACGUAAGUAA CAGUUUGAAU GGUUGAAGUA U-3&amp;prime;-OH</td><td>For self-replicator ribozyme reactions (section 6)</td></tr><tr><td>Self-replicator substrate B, transcribed</td><td>prepared in house&lt;sup&gt;14&lt;/sup&gt;</td><td>5&amp;prime;-pppGAGACCGCAA CUUA-3&amp;prime;</td><td>For self-replicator ribozyme reactions (section 6)</td></tr><tr><td>Small Drying Traps, 4 &amp;Aring; molecular sieves</td><td>ChemGenes</td><td>DMT-1975</td><td>Drying traps for DNA/RNA synthesizer phosphoramidites and triphosphorylation reagents (sections 1&amp;ndash;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&amp;ndash;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&amp;ndash;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&amp;ndash;7)</td></tr><tr><td>Tetrabutylammonium Fluoride, 1.0 M solution in Tetrahydrofuran</td><td>Aldrich</td><td>216143</td><td>For removing 2&amp;prime;-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&amp;ndash;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&amp;ndash;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&amp;ndash;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 ...

chemical-triphosphorylation-of-oligonucleotides

Research

JoVE Journal

Bioengineering

3.2K Views.  The Salk Institute. Oligonucleotide 5′-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′-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

Video: Chemical Triphosphorylation of Oligonucleotides

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

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.

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.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Chemistry

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Genetics

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Biochemistry

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to ...

Phosphodiester Linkages

<h4>Overview</h4>
Phosphodiester bond forms when a phosphoric acid molecule (H3PO4) links with two hydroxyl groups (&#8211;OH) of two other molecules, forming two ester bonds. Two water molecules are released in this process. The phosphodiester bond is commonly found in nucleic acids (DNA and RNA) and plays a critical role in their structure and function.
<h4>Phosphodiester Bonds Link Nucleotides Together</h4>
DNA and RNA are polynucleotides or long chains of nucleotides that are linked together. A nucleotide is composed of a nitrogenous base (adenine, guanine, thymine, cytosine, or uracil), a pentose sugar, and a phosphate molecule (PO3&#8722;4). In a polynucleotide chain, nucleotides are linked together by phosphodiester bonds, where each phosphate group forms two ester bonds with its neighboring sugar molecules. The first ester bond already exists between the phosphate group and the 5&#39; carbon of the pentose sugar of the nucleotide. The second ester bond is formed between the hydroxyl group (&#8211;OH) attached to the 3&#8217; carbon of the preceding sugar molecule and the phosphate group. The formation of each ester bond removes one water molecule. A class of enzymes called polymerases to&#160;catalyze, or accelerate, the formation of phosphodiester bonds.
Phosphodiester bonds in a polynucleotide chain form an alternating pattern of sugar and phosphate residues called the sugar-phosphate backbone. Phosphodiester bonds impart directionality to a polynucleotide chain. The polynucleotide chain has a free 5&#39; phosphate group at one end and a free 3&#39; hydroxyl group at the other. These ends are called the 5&#39; end and the 3&#39; end, respectively. The directionality of nucleic acids is essential for DNA replication and RNA synthesis.

Phosphodiester Linkages and Nucleotides

Overview
Phosphodiester bond forms when a phosphoric acid molecule (H3PO4) links with two hydroxyl groups (&#8211;OH) of two other molecules, forming two ...

Chemical Triphosphorylation of Oligonucleotides

Summary

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Chapters in this video

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

Oligopeptide Competition Assay for Phosphorylation Site Determination

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Phosphodiester Linkages