This work was supported by the Swiss National Science Foundation (Grants n&deg; PZ00P2_126430/1 and PZ00P2_144595). Prof. C. Leumann is gratefully acknowledged for providing the lab space and equipment, as well as for his constant support. Ms. Sue Knecht is acknowledged for fruitful discussions.

1. Synthesis of the Modified Nucleoside Triphosphates
The synthetic approach chosen follows the procedure developed by Ludwig and Eckstein since this method is generally reliable and leads to very few side-products (Figure 1A)39.
<ol>
	<li>Coevaporate the suitably 3&#8217;-OAc-protected nucleoside (&#160;typically&#160;0.1 mmol) twice with anhydrous pyridine (2 ml) and then dry under vacuum overnight. At the same time, dry tributylammonium pyrophosphate (0.13 mmol) under vacuum overnight.</li>
	<li>Dissolve the nucleoside in a minimum of dry pyridine (0.2 ml) and add dry dioxane (0.4 ml) as a cosolvent. Finally, add 2-chloro-1,3,2-benzodioxaphosphorin-4-one (0.11 mmol) and allow to react at room temperature for 45 min. 
	Note: It is noteworthy that particular care should be taken with the 2-chloro-1,3,2-benzodioxaphosphorin-4-one reagent. Indeed, even storage of this reagent under an inert gas atmosphere is not sufficient to prevent its decomposition. Thus, the white solid that is formed in this decomposition can and should be scraped off prior to use.</li>
	<li>Prepare a solution of tributylammonium pyrophosphate in dry DMF (0.17 ml) and freshly distilled tributylamine (58 &#181;l; never add molecular sieves). Add the resulting solution to the reaction mixture (a white precipitate appears but quickly disappears) and allow to react at room temperature for 45 min.</li>
	<li>Prepare a solution of iodine (0.16 mmol) in pyridine (0.98 ml) and H2O (20 &#181;l) and add to the reaction mixture in order to oxidize the P&#945;(III) center. Allow the resulting dark solution to stir at room temperature for 30 min.</li>
	<li>Use a 10% aqueous solution of NaS2O3 to quench the excess I2. Remove the solvent under vacuum (the temperature of the water bath of the rotary evaporator must be kept below 30 &#176;C). Add H2O (5 ml) and allow the mixture to stand at room temperature for 30 min to hydrolyze the cyclic triphosphate moiety.</li>
	<li>At this stage, the protecting groups are usually removed (i.e. the 3&#39;-OAc and the groups on the side chains of the nucleobase). Consequently, add NH4OH (30% in H2O, 10 ml) to the crude and stir for 1.5 hr at room temperature, and remove the solvent under vacuum.</li>
	<li>Add 2 ml of H2O and split into 2 tubes. Add 12 ml of a 2% solution of NaClO4 in acetone and centrifuge (1,000 x g) for 30 min. This procedure is repeated one more time. This precipitation allows for the separation of the solvents and reagents used in the synthesis from the triphosphate (which will precipitate), thus simplifying the ensuing RP-HPLC purification substantially.</li>
	<li>After air-drying of the oily residue, record a 31P-NMR spectrum of the crude (following standard procedures, see also List of Materials and Figure 3) and dissolve in 4 ml H2O. 
	Note: This synthetic procedure mainly focuses on the generation of modified deoxynucleoside triphosphates, but a very similar procedure can be applied for their RNA counterparts (by simply using 2&#39;,3&#39;-bis-O-acetylated precursors).</li>
</ol>
2. HPLC Purification of the Modified Nucleoside Triphosphates
<ol>
	<li>Preparation of a 1 M stock solution of triethylammonium bicarbonate (TEAB):40
	<ol>
		<li>Prepare a solution of 1 mole of triethylamine (139 ml) in &#8776; 600 ml of distilled and filtered H2O.</li>
		<li>Bubble CO2 (via dry ice and a gas bubbler) into the solution under vigorous stirring until a pH of 7.6 is reached (this will take at least 10 hr). This stock solution can be stored in the fridge for up to one month.</li>
	</ol>
	</li>
	<li>Purification:
	<ol>
		<li>Prepare two buffer solutions from the 1 M stock: 2 L of 50 mM TEAB in ultrapure water (eluent A) and 1 L of 50 mM TEAB in 50% MeCN (eluent B). Degas both eluents under vacuum and stirring for 20 min (careful attention is required so as not to alter the pH by removing CO2 during degassing).</li>
		<li>Prepare an analytical sample by dissolving 10 &#181;l of the crude triphosphate in 300 &#181;l distilled H2O and inject into an HPLC system equipped with a semi-preparative RP column (C18) and using a gradient ranging from 0-100% B in 40 min (Figure 4). Adjust the HPLC program according to the Rt of the triphosphate (which is usually the main peak on the chromatogram) and the diphosphate (which has a slightly lower Rt). Purify the crude mixture using these conditions and by removing early fractions that might contain some diphosphate.</li>
		<li>Combine all the fractions that contain the product and freeze-dry (which is preferred to evaporation on the rotary evaporator in order to minimize hydrolysis to the undesired diphosphate). Coevaporate several times with ultrapure water (to remove remnant triethylamine from the eluents). Assess the purity of the triphosphate by NMR (both 1H and 31P NMR) and MALDI-TOF by application of standard protocols (see Figure 5). Typical yields obtained by application of this protocol typically lie in the range of 30-70%, depending on the substrate.</li>
	</ol>
	</li>
</ol>
3. Primer Extension Reactions and TdT Polymerization
<ol>
	<li>5&#39;-end labeling of the primer
	<ol>
		<li>Mix 30 pmol of the appropriate primer (e.g. P1, List of Materials) with 4 &#181;l of 10x Polynucleotide kinase (PNK) buffer, 3 &#181;l of &#947;-[32P]-ATP, 1 &#181;l of PNK, and ddH2O (for a total reaction volume of 40 &#181;l). Incubate the reaction mixture at 37 &#176;C for 30 min and then heat-deactivate the kinase (10 min at 70 &#176;C).</li>
		<li>During the labeling reaction prepare a G10 Sephadex column by plugging a 1 ml pipette tip with silanized glass wool and filling the tip with G10 solution (10% in ddH2O, autoclaved). Spin down and wash three times with 500 &#181;l ddH2O and one final wash with 50 &#181;l ddH2O. Run the reaction mixture through the G10 (to remove the free radioactive label).</li>
		<li>Gel purify (PAGE 20%) the radiolabeled primer and recover by application of the crush and soak method (i.e. elution with 500 &#181;l of an aqueous solution containing 1% LiClO4 and 1 mM NEt3 (pH 8) at 72 &#176;C for 15 min). Ethanol precipitate and G10 desalt the eluted oligonucleotide.</li>
	</ol>
	</li>
	<li>Primer extension reaction
	<ol>
		<li>Anneal 1 pmol of radiolabeled primer P1, 10 pmol primer P1, and 10 pmol of template T1 (List of Materials) in 10x reaction buffer (provided by the supplier of the polymerase to be used) by placing the tube in hot (90-95 &#176;C) water and by allowing to gradually cool down to room temperature (over 45 min).</li>
		<li>Put the tube on ice and add (in turn) the dNTP cocktail (containing both the modified and the natural triphosphates, 100 &#181;M final concentration) and 1 U of the DNA polymerase (Vent (exo-), Klenow, Pwo or 9&#176;Nm) and complete with water (for a total reaction volume of 20 &#181;l). Incubate at the optimal working temperature of the enzyme (e.g. 60 &#176;C for 30 min when Vent (exo-) is being used).</li>
		<li>Add 20 &#181;l of stop solution (formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mM), bromophenol blue (0.1%), xylene cyanol (0.1 %)), heat the samples (95 &#176;C, 5 min), cool down (0 &#176;C), and resolve by denaturing polyacrylamide gel electrophoresis. Visualize by phosphorimager.</li>
	</ol>
	</li>
	<li>TdT polymerization
	<ol>
		<li>Dilute 7 pmol of single-stranded, unlabeled primer P2 (List of Materials), 1 pmol of radiolabeled primer in 1 &#181;l TdT buffer 10x.</li>
		<li>Put the tube on ice and add (in turn) the dNTP cocktail (at an adequate concentration comprised between 10 and 200 &#181;M) and the TdT polymerase (4 U). Incubate at 37 &#176;C for 1 hr. Add 10 &#181;l of the stop solution, heat the samples (95 &#176;C, 5 min), cool down (0 &#176;C), and resolve by denaturing polyacrylamide gel electrophoresis (PAGE 15%). Visualize by phosphorimager.</li>
	</ol>
	</li>
</ol>
4. PCR with Modified Nucleoside Triphosphates
<ol>
	<li>Mix 8 pmol of both the forward and reverse primers with 0.5 pmol of the template to be amplified. Add 10x reaction buffer and the dNTP cocktail (for a 200 &#181;M final concentration) and put the tube on ice. Add 1 U of the DNA polymerase and complete with H2O to reach a final volume of 20 &#181;l. Transfer the mixture into a PCR vial and carry out 30 PCR cycles.</li>
	<li>Dilute 6 &#181;l with 6 &#181;l of 2x sucrose loading buffer and resolve by agarose 2% (stained with ethidium bromide) electrophoresis. Visualize by phosphorimager.</li>
</ol>

No conflicts of interest declared.

The inclusion of modifications into nucleic acids is of interest for numerous practical applications including the development of antisense and antigene agents42,43, labeling and functional tagging of oligonucleotides41, and in efforts to expand the genetic alphabet44-46. Chemical alterations and functional groups are usually introduced into nucleic acids by application of standard and automated solid-phase synthesis protocols. However, the phosphoramidite building blocks need to be resilient to the rather harsh conditions imposed by this methodology, which in turn imposes a severe restriction onto the nature of the chemical functionality47. Instead, the enzyme-mediated polymerization of modified nucleoside triphosphates allows for the introduction of a broader range of functionalities, since the sole restriction is that they act as substrates for polymerases1,2. Even though there is a noticeable lack of generally applicable methodology, reliable and robust synthetic and analytical methodologies have been developed for the synthesis of modified dNTPs. Moreover, due to their inherent nature, modified triphosphates are rather sensitive to different external conditions (e.g. pH, temperature) and thus, a detailed protocol for their synthesis and characterization is highly beneficial.
The workflow presented herein includes the chemical synthesis of modified dNTPs, RP-HPLC purification, NMR analysis, and enzymatic assays for the biochemical characterization of these nucleoside analogues. The most critical steps for a successful synthesis and characterization of modified dNTPs are the analysis of the crude product (by NMR), the thorough HPLC purification, the analysis of the purified material, and choice of an appropriate DNA (RNA) polymerase.
For the synthesis of the modified dNTPs we applied the method developed by Ludwig and Eckstein39, since fewer by-products are formed as compared to other procedures, albeit at the expense of a slightly longer synthetic route. Furthermore, the RP-HPLC purification certainly represents the pivotal step of the entire synthetic route since it will allow for the separation of the nucleoside diphosphates (dNDPs) which often strongly inhibit DNA and RNA polymerases48. After achieving the synthesis and purification, the purity of the resulting triphosphate needs to be assessed both by NMR and MALDI-TOF to ensure that no polymerase-inhibiting diphosphate is present.
The incorporation assay shown in Figure 6 clearly underscores the usefulness of this approach. Indeed, all of the dNTPs employed in this representative example reveal to be good substrates for the DNA polymerase (in this particular case Vent(exo-)) since no faster running bands corresponding to smaller fragments could be observed. Besides, the enzyme tolerates two substrates adorned with carboxylic acid residues (dUtPTP (3) and dCValTP (7)) and the polymerization of both dNTPs results in an oligonucleotide bearing not less than 39 additional negative charges. Another striking feature is that the bands resulting from the incorporation of modified dNTPs often display slower electrophoretic mobilities than the natural controls (compare e.g. lanes 11 and 12). Thus, primer extension reactions represent a powerful and yet simple way to assess the substrate acceptance of modified dNTPs.
Moreover, the TdT-mediated polymerization of triphosphates on the 3&#39;-termini of single stranded oligonucleotides is an alluring strategy for the generation of highly functionalized nucleic acids49-52.&#160;The representative example shown in Figure 7 clearly demonstrates the procedure for selecting the functionalities that are tolerated by the Co2+-dependent TdT53. Indeed, dUcPTP (1) and dUFPTP (3), which are equipped with the proteinogenic amino acid L-proline and the dipeptide &#945;-Phe-Pro, respectively, are the best substrates for the TdT and gave rise to tailing efficiencies that compare favorably to the unmodified dTTP control, even at concentrations as low as 10 &#181;M (lanes 1 and 4). Surprisingly, analogue dUtPTP (2) where the proline residue is connected to the linker arm in trans compared to the free carboxylic acid, is not as good a substrate as its cis counterpart since polydisperse longer-sized oligonucleotides are only observed at higher dNTP concentrations (&#62; 100 &#181;M, lane 2). Furthermore, the sulfonamide-modified dNTP 5 is a moderate substrate for TdT and comparable to dUtPTP (2) (lane 3). In addition, dUBpuTP (4), which bears a strong hydrogen-bond donating motif, is a rather poor substrate for TdT and the polymerization reaction seems to be indifferent to the concentration of the modified dNTP. Thus, the following order of substrate acceptance can be construed from Figure 7: dUcPTP (1), dUFPTP (3)&#62; dUtPTP (2), dUBsTP (5) &#62; dUBpuTP (4).
Finally, the procedure described for polymerization reactions under PCR conditions using the modified analogues is rather similar to that involving natural dNTPs. However, the compatibility of modified dNTPs with polymerases under these conditions is of crucial importance for the generation of high-density functionalized nucleic acids7, especially with regards to their use in in vitro selection experiments.
In summary, the method for the synthesis and characterization of modified nucleoside triphosphates was emphasized and the establishment of such a protocol will certainly help in the development and crafting of novel analogues. Concomitantly, the emergence of such novel dNTPs will facilitate the generation of functionalized oligonucleotides; particularly, catalytic nucleic acids.

5&#39;-Nucleoside triphosphates ((d)NTPs) represent a class of vital biomolecules that are involved in countless processes and functions ranging from being the universal currency of energy to regulators of cell metabolism. In addition to their role in these fundamental biological transformations, their modified counterparts have advanced as a versatile and mild platform for the introduction of functional groups into oligonucleotides, a methodology that nicely complements the automated solid-phase synthesis that is usually applied1,2. Indeed, provided the (d)NTPs can act as substrates for RNA and DNA polymerases3, a wealth of functional groups including amino acids4-13, boronic acids14,15, nornbornene16, diamondoid-like residues17, side-chains for organocatalysis18, bile acids19, and even oligonucleotides20 can be introduced into oligonucleotides.
Beyond representing a convenient vector for the functionalization of nucleic acids, modified dNTPs can be engaged in SELEX and other related combinatorial methods of in vitro selection for the generation of modified catalytic nucleic acids21-30 and aptamers for various practical applications10,31-36. The additional side-chains that are introduced by the polymerization of the modified dNTPs are thought to increase the chemical space that can be explored during a selection experiment and supplement the rather poor functional arsenal of nucleic acids37. However, despite these attractive traits and the recent progress made in the development of both synthetic and analytical methods, no universally applicable and high-yielding procedure exists for the crafting of modified nucleoside triphosphates2,38.
The aim of this present protocol is to shed light into the (sometimes) intricate procedures leading to the synthesis and biochemical characterization of these activated building blocks (Figure 1B). Special emphasis will be given on all the synthetic details that often are difficult to find or are absent in experimental sections but are yet crucial for the successful completion of the synthetic pathway leading to the isolation of pure (d)NTPs (Figure 1).

<table><tbody><tr><td>Tributylammonium pyrophosphate&amp;nbsp;</td><td>Sigma Aldrich</td><td>P8533</td><td>Hygroscopic solid, keep under Ar</td></tr><tr><td>2-Chloro-1,3,2-benzodioxaphosphorin-4-one&amp;nbsp;</td><td>Sigma Aldrich</td><td>324124</td><td>Moisture sensitive</td></tr><tr><td>Pyridine</td><td>Sigma Aldrich</td><td>82704</td><td>Under molecular sieves</td></tr><tr><td>Dioxane</td><td>Sigma Aldrich</td><td>296309</td><td>Under molecular sieves</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Sigma Aldrich</td><td>40248</td><td>Under molecular sieves</td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td>HPLC grade</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>90342</td><td></td></tr><tr><td>Tributylamine</td><td>Sigma Aldrich</td><td>90781</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Milli-Q</td><td></td><td>deionized and purified water, autoclaved in the presence of Diethylpyrocarbonate (DEPC)</td></tr><tr><td>Diethylpyrocarbonate (DEPC)</td><td>Sigma Aldrich</td><td>159220</td><td></td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;O</td><td>Cambridge Isotope Laboratories, Inc.</td><td>DLM-4-25</td><td></td></tr><tr><td>&amp;#947;-[&lt;sup&gt;32&lt;/sup&gt;P]-ATP</td><td>Hartmann Analytics</td><td>FP-301</td><td></td></tr><tr><td>Natural dNTPs</td><td>Promega</td><td>U1420</td><td></td></tr><tr><td>Vent (exo&lt;sup&gt;-&lt;/sup&gt;) DNA polymerase</td><td>NEB</td><td>M0257S</td><td></td></tr><tr><td>DNA polymerase I, Large (Klenow) Fragment</td><td>NEB</td><td>MO210S</td><td></td></tr><tr><td>9&amp;deg;&lt;em&gt;N&lt;sub&gt;m&lt;/sub&gt;&lt;/em&gt; DNA polymerase</td><td>NEB</td><td>MO260S</td><td></td></tr><tr><td>Terminal deoxynucleotidyl Transferase (TdT)</td><td>Promega</td><td>M828A</td><td></td></tr><tr><td>Pwo DNA polymerase</td><td>Peqlab</td><td>01 01 5010</td><td></td></tr><tr><td>T4 PNK</td><td>Thermo Scientific</td><td>EK0032</td><td></td></tr><tr><td>Acrylamide/bisacrylamide (19:1, 40%)</td><td>Serva</td><td>10679.01</td><td></td></tr><tr><td>Agarose</td><td>Apollo Scientific</td><td>BIA1177</td><td></td></tr><tr><td>G10 Sephadex</td><td>Sigma</td><td>G10120</td><td></td></tr><tr><td>Urea</td><td>Apollo Scientific</td><td>BIU4110</td><td></td></tr><tr><td>Jupiter semipreparative RP-HPLC column (5&amp;#956; C18 300 &amp;Aring;)</td><td>Phenomenex</td><td></td><td></td></tr><tr><td>Gene Q Thermal Cycler</td><td>Bioconcept</td><td>BYQ6042E</td><td></td></tr><tr><td>PCR vials</td><td>Bioconcept</td><td>3220-00</td><td></td></tr><tr><td>HPLC system</td><td>Amersham Pharmacia Biotech</td><td></td><td>&amp;Auml;kta basic 10/100</td></tr><tr><td>All oligonucleotides were purchased from Microsynth and purified by PAGE</td><td></td><td></td><td></td></tr><tr><td>5&amp;#039;-CAAGGACAAAATAC&lt;br /&gt;
CTGTATTCCTT &lt;strong&gt;P1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;#039;-GACATCATGAGAGA&lt;br /&gt;
CATCGCCTCTGGGCTA&lt;br /&gt;
ATAGGACTACTTCTAAT&lt;br /&gt;
CTGTAAGAGCAGATCC&lt;br /&gt;
CTGGACAGGCAAGGAA&lt;br /&gt;
TACAGGTATTTTGTCCTTG &lt;strong&gt;T1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;#039;-GAATTCGATATCAAG &lt;strong&gt;P2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td colspan="4">More information on experimental procedures and equipment can be found in the following articles:&lt;br /&gt;
&lt;em&gt;Chem. Eur. J.&lt;/em&gt; &lt;strong&gt;2012&lt;/strong&gt;, 18, 13320-13330&lt;br /&gt;
&lt;em&gt;Org. Biomol. Chem.&lt;/em&gt; &lt;strong&gt;2013&lt;/strong&gt;, DOI: 10.1039/C3OB40842F</td></tr></tbody></table>

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.

Modified nucleoside triphosphates are alluring synthetic targets since they allow for the facile introduction of an vast array of functional groups into nucleic acids41. However, the isolation and characterization of these activated building blocks is often revealed to be arduous. Consequently, the results shown herein are thought to provide a helping hand to follow the various steps within the aforementioned synthetic and biochemical procedures (Figure 1B).
In particular, Figure 3 shows a typical crude 31P-NMR spectrum of a modified dNTP (in this particular case, dUBpuTP (4)18, Figure 2), where the characteristic signals of the phosphorous centers can be observed (i.e. a doublet at -5.02 (P&#947;), a doublet at -10.44 (P&#945;), and a triplet at -20.55 (P&#946;) ppm). In addition, signals stemming for the diphosphate (two doublets at -4.84 and -10.63 ppm) and monophosphate (singlet at -0.18 ppm) side-products along with higher phosphates (signals at -21.02 and -23.19 ppm) are always observed at this stage. A first RP-HPLC analysis of the crude mixture is shown on Figure 4 (for dUBpuTP (4)) where the main peak (Rt = 30.78 min) corresponds to the 5&#39;-triphosphate, while the main byproduct, the 5&#39;-diphosphate, displays a slightly lower retention time (Rt = 30.03 min). Finally, after a thorough RP-HPLC purification, the modified dNTP needs to be characterized by NMR and MALDI-TOF (Figure 5). Both the 1H-NMR and 31P-NMR spectra are crucial for assessing the purity of the modified dNTPs, since the presence of undesired di- and mono-phosphates gives distinctive signals.
After establishing the purity of the nucleoside analogue and assessing the concentration of the stock solution either by mass or by UV-spectroscopy, the modified dNTP can be used in primer extension reactions in order to assess its substrate acceptance capacity by various polymerases. Figure 6 illustrates the outcome of primer extension reactions with dUtPTP (2), dAHsTP (6), and dCValTP (7) used either as lone modifications (lanes 5-7), as combinations of two modified dNTPs (lanes 8-10), or together along with the lone natural dGTP (lane 11).
Finally, Figure 7 shows representative TdT-mediated polymerization reactions with different modified dUTP analogues. In this context, dUcPTP (1) and dUFPTP (3) are the best substrates for TdT (lanes 1 and 4) since the tailing efficiencies are comparable or exceed those of the natural dTTP (lane 6). Instead, dUBpuTP (4) (lane 5) is a rather poor substrate in this context since little polydisperse longer-sized oligonucleotides can be observed.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig1highres.jpg" src="/files/ftp_upload/51385/51385fig1.jpg" width="600px" /> 
Figure 1. A) Ludwig-Eckstein approach for the synthesis of (base) modified nucleoside triphosphates39. B) Schematic representation of all the steps required for the synthesis and biochemical characterization of modified dNTPs prior to their use in applications such as SELEX. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig2highres.jpg" src="/files/ftp_upload/51385/51385fig2.jpg" width="600px" /> 
Figure 2. Chemical structures of the modified nucleoside triphosphates: dUcPTP (1), dUtPTP (2), dUFPTP (3), dUBpuTP (4), dUBsTP (5),18 dAHsTP (6), and dCValTP (7)&#160;13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig3highres.jpg" src="/files/ftp_upload/51385/51385fig3.jpg" width="600px" /> 
Figure 3. 31P-NMR spectrum (121.4 MHz, D2O) of the crude reaction mixture of dUBpuTP (4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig4highres.jpg" src="/files/ftp_upload/51385/51385fig4.jpg" width="600px" /> 
Figure 4. RP-HPLC profile of crude dUBpuTP (4): 0-100% eluent B in 40 min, flow rate: 3.5 ml/min&#160;(eluent A: 50 mM TEAB in H2O; eluent B: 50 mM TEAB in H2O/CH3CN (1/1)). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig5highres.jpg" src="/files/ftp_upload/51385/51385fig5.jpg" width="600px" /> 
Figure 5. Characterization of the modified nucleoside triphosphate dUBsTP (5): A) 31P-NMR spectrum (121.4 MHz, D2O, 128 scans);18 B) 1H-NMR spectrum (300 MHz, D2O, 128 scans); C) MALDI-TOF spectrum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig6highres.jpg" src="/files/ftp_upload/51385/51385fig6.jpg" width="600px" /> 
Figure 6. Representative gel image (PAGE 15%) of primer extension reactions with various base modified dNTP analogues. Lane 1: primer; lane 2: natural dNTPs without dUTP; lane 3: natural dNTPs without dATP; lane 4: natural dNTPs without dCTP; lane 5: dUtPTP (2); lane 6: dAHsTP (6); lane 7: dCValTP (7); lane 8: dAHsTP (6) and dUtPTP (2); lane 9: dAHsTP (6) and dCValTP (7); lane 10: dUtPTP (2) and dCValTP (7); lane 11: dUtPTP (2), dAHsTP (6), and dCValTP (7); lane 12: natural dNTPs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig6highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51385/51385fig7highres.jpg" src="/files/ftp_upload/51385/51385fig7.jpg" width="600px" /> 
Figure 7. Gel image (PAGE 20%) of the TdT polymerization reactions with various base modified dUTP analogues. Lane 1: dUcPTP (1); lane 2: dUtPTP (2); lane 3: dUBsTP (5); lane 4: dUFPTP (3); lane 5: dUBpuTP (4); lane 6: dTTP; lane 7: primer. Concentrations: 10 &#181;M, 25 &#181;M, 50 &#181;M, 75 &#181;M, and 100 &#181;M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51385/51385fig7highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Nucleoside Triphosphates - From Synthesis to Biochemical Characterization at JoVE.com

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

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 ...

nucleoside-triphosphates-from-synthesis-to-biochemical

Nanyang Technological University

Research

JoVE Journal

Chemistry

17.0K Views.  University of Bern. 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.

Video: Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

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

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

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 ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Biochemistry

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

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.

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 ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Summary

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Chapters in this video

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Synthesis and Characterization of Functionalized Metal-organic Frameworks

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

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

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

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

NMR-Based Activity Assays for Determining Compound Inhibition, IC₅₀ Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Chemical Triphosphorylation of Oligonucleotides