Preparation of this manuscript was supported via start-up funds from the University of Colorado Denver (JMRE). AF would like to acknowledge support from a Research and Creative Activities award (RaCAS, CU Denver). Funding from the Office of Research Services, University of Colorado Denver to cover publication charges is acknowledged. We would like to thank lab members Ms. Cassandra Herbert and Mr. Yannick K. Dzowo for their contributions in the video portion.

1. Solid-phase Synthesis of RNA Oligonucleotides
<ol>
	<li>Preparation of solutions containing each phosphoramidite (Table 1).
	<ol>
		<li>Count the number of nucleotides and fit in the n+1 equation (where n = number of nucleotides) corresponding to each base and fill in the table with the missing values. Volume = 0.15 mL per nucleotide at a concentration of 0.1 M, dissolved in anhydrous acetonitrile.</li>
		<li>Weigh each phosphoramidite into oven-dried 10 mL amber bottles (Septum Top Amber 394 Amidite 13 mm ID x 20 mm OD Top) and place immediately under vacuum using a desiccator containing a drying agent.</li>
		<li>Fill the desiccator with dry argon, remove the bottle and dilute immediately with the corresponding amount of acetonitrile (anhydrous) before securing onto the instrument. 
		NOTE: Make sure to use gas-tight syringes and keep an anhydrous atmosphere at all times.</li>
		<li>Remove the septa from the bottle and place on the machine, via a bottle change function. 
		NOTE: This process requires an additional 0.15 mL (hence the excess volume in the n+1 equation above) of each solution to prime the line prior to synthesis.</li>
	</ol>
	</li>
	<li>Set-up of sequence and solid phase synthesis using the software (Figure 3).
	<ol>
		<li>Click on the software icon (e.g., OligoNet 1.0.1) and select Instrument name&#62;OK. Create a new synthesis by File&#62;New Synthesis&#62;Order.</li>
		<li>Fill-in: Date; RunID; select instrument; sequence name; sequence (5&#39;-to-3&#39; end). Under |cycle, assign the previously created method (Figure 3). Choose the end procedure, (leaves oligonucleotide bound to the CPG resin)&#62;manual.</li>
		<li>Select DMT Off &#62;(TCA treatment in the last step to obtain a 5&#39;-OH group at the end of the synthesis); save File&#62;Save as and provide a name for the experiment.</li>
		<li>Send the file to begin synthesis Order&#62;Send order to synthesizer and select column 1-4. Open the synthesizer window and select trityl monitor|choose function|trity&#62;monitor every step.</li>
		<li>Repeat the steps as necessary depending on the number of oligonucleotides to be synthesized at once (note that all orders must have the same method, i.e., same coupling times and sequence of events).</li>
		<li>Begin the synthesis Synthesizer&#62;Prepare to start. Place the columns with the desired 3&#39;-end on the positions indicated on the instrument. On the instrument click Start&#62;No (for ABI preparation).</li>
		<li>Once the synthesis is complete, remove the column from the instrument and place in a round bottom flask followed by drying under reduced pressure for about 0.5 h. 
		NOTE: It is recommended to check that a deep orange color is observed in random couplings (steps 1.2.4 - 1.2.7) to avoid potential errors from the trityl monitor function.</li>
	</ol>
	</li>
	<li>Deprotection and purification of RNA oligonucleotides.
	<ol>
		<li>Open the column by twisting the black cap and transfer all (or half, depending on the need or purpose) of the white resin into a 1.6 mL centrifuge tube. Add 0.5 mL of a methylamine (40% in water)/ammonia (40% in water) at 1:1.</li>
		<li>Secure the centrifuge tube cap with parafilm and, using a heat-block, heat to 60 &#176;C for 1.5 h. 
		NOTE: A heavy object can be placed on top of the tube to ensure that the ammonia concentration remains constant inside the reaction tube.</li>
		<li>Remove from heat block and cool slowly to room temperature. Briefly centrifuge the sample to spin down contents, then transfer the supernatant to a new centrifuge tube.</li>
		<li>Freeze the samples by submerging tubes in liquid nitrogen (or dry ice/ethanol bath) and concentrate to dryness while spinning in a centrifuge under reduced pressure.</li>
		<li>Re-suspend solids in 0.4 mL of a triethylamine/N-methylpyrrolidinone/triethylamine-trihydrofluoride (2:2:3 ratio) solution. Heat to 60 &#176;C for 1.5 h followed by slow cooling to room temperature.</li>
		<li>Add 0.04 mL of a NaOAc solution (3M, pH 5.5 - adjusted with HCl) followed by gentle mixing with a pipette tip. Add ethanol (1 mL) and cool to ca. -70 &#176;C (dry ice/ethanol bath) for 15 min.</li>
		<li>Centrifuge at 15,000 rpm and 4 &#176;C for 10 min. Use a pipette to extract the aliquot and dry the resulting pellet under reduced pressure.</li>
		<li>Re-suspend the obtained solid in loading buffer (0.2 mL, 90% aq. formamide, 1 mM EDTA) and mix until mixture is homogeneous.</li>
		<li>Load suspension onto a previously prepared polyacrylamide gel (20% denaturing, dimensions of well: 30 cm x 1 mm)22.</li>
		<li>Apply a current through the gel until the bromophenol blue marker is located between 1/2-2/3 down the gel (gel dimensions: ~ 21.5 cm x 30 cm).</li>
		<li>Remove the gel from the stand and transfer the gel contents from the glass to plastic wrap (covered on both sides), and place over a thin layer chromatography (TLC) plate covered with silica (containing fluorescent dye at 254 nm) to visualize the bands using a UV-lamp (&#955;max = 254 nm).</li>
		<li>Use a marker to delineate the position where the upper band is located and cut it using a new razor blade. Place gel containing the RNA (without plastic wrap) into a 50-mL conical tube and crush to small pieces using a glass rod.</li>
		<li>Suspend the gel residues into a NaCl aq. solution (200 mM NaCl&#160;and 1 mM EDTA) and shake the suspension at 37 &#176;C for 12 h. Centrifuge the conical tube for 10 min.</li>
		<li>Desalt by using a reverse-phase C18 cartridge.
		<ol>
			<li>Prepare the cartridge using a 10-mL syringe by washing with: 
			Acetonitrile (10 mL) 
			H2O (twice, 10 mL) 
			5 mM NH4OAc aq. solution (3 mL) 
			Solution containing RNA, taking care not to pour any of the gel residuals.</li>
			<li>Wash with H2O (three times, 10 mL).</li>
			<li>Elute from the column using a 60% aq. methanol solution (3 mL)</li>
		</ol>
		</li>
		<li>Concentrate under reduced pressure and re-dissolve in RNase-free water (0.3 mL)</li>
		<li>Prepare a dilute solution (10 &#181;L) and deposit 1 &#181;L on the UV-vis instrument (e.g., Nanodrop) to measure the UV-vis spectrum (200-450 nm).</li>
		<li>Use Beer Lambert&#39;s Law to determine the concentration of the obtained solution: 
		<img alt="Equation 1" src="/files/ftp_upload/56189/56189eq1.jpg" /> 
		where A = obtained absorbance; &#949; = calculated molar extinction coefficient; c = concentration, and l = 0.1 for a path length of 1 mm.</li>
		<li>Calculate the molar extinction coefficients for the oligonucleotides; calculated here with an on-line calculator that uses DINAMelt software (http://unafold.ra.albany.edu/?q=dinamelt)23</li>
	</ol>
	</li>
	<li>Concentration determination and characterization via MALDI-TOF.
	<ol>
		<li>Load samples onto a MALDI plate using a pipette tip loaded with a C18 tip to desalt, and spot each oligonucleotide.
		<ol>
			<li>Wash the tip with 50% acetonitrile (10 &#181;L x 2). Equilibrate the tip with 0.1% trifluoroacetic acid (TFA; 10 &#181;L x 2). Load tip with sample (typically 100-150 pmol).</li>
			<li>Wash the tip with 0.1% TFA (10 &#181;L x 2), and then with water (10 &#181;L x 2).</li>
			<li>Elute the sample into a solution containing the desired matrix; we used a solution of: 10 &#181;L 25 mM-2,4,6-trihydroxyacetophenone monohydrate (THAP), 10 mM ammonium citrate, and 300 mM ammonium fluoride in 50% acetonitrile.</li>
			<li>Spot directly onto the MALDI plate by depositing 0.9 &#181;L (followed by air drying) and repeating the procedure as necessary. 
			NOTE: All spectra were obtained on the reflector positive-mode.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. RNA Structure Analysis via CD
<ol>
	<li>Preparation of solutions for CD.
	<ol>
		<li>Prepare 0.25 mL containing the modified RNA [3 &#181;M], NaCl [10 mM], sodium phosphate buffer [10 mM, pH 7.2], and MgCl2 [5 mM]. The sample is ready for analysis as is, particularly if it represents a unimolecular transition.</li>
		<li>If the goal is to analyze duplex structures or bimolecular transitions, add complement (1 molar equivalent, if applicable) at this time, or continue with the step below.</li>
		<li>Place the sample on a heat-block, pre-heated to 90 &#176;C, and turn-off the heat to control slow cooling to room temperature (typically 2 - 4 h).</li>
	</ol>
	</li>
	<li>Spectra acquisition
	<ol>
		<li>Prepare a blank solution (NaCl 10 mM, sodium phosphate 10 mM pH 7.3, MgCl2 5 mM), transfer to a micro-cuvette (1 cm path length, 250 &#181;L minimum volume), and place on a holder position of the sample changer within the instrument.</li>
		<li>Transfer the sample containing RNA into another micro-cuvette and position in sample changer. If measuring a thermal denaturation transition, carefully add a bed of oil without disrupting the aqueous layer and secure the cap of cuvette using a piece of Teflon tape.</li>
		<li>Open the nitrogen tank to provide a flow that moves the air floater located on the instrument to ca. 40. Turn-on the cooler.</li>
		<li>Turn-on the instrument and open the software Spectra Manager icon, then open the acquisition window Spectra Measurement.</li>
		<li>Purge the instrument with nitrogen for 5 min before acquisition.</li>
		<li>Acquire the spectra using the following parameters Measure&#62;Parameters and adjust the parameters as follows:
		<ol>
			<li>Under General, select: Scan 200-350 nm; Channels CD and HT: Data pitch at 0.1 nm; scanning speed at 100 nm/min; band width at 1 nm; number of accumulations at 5.</li>
			<li>Under cell unit, choose 20 &#176;C. Under control, select Shutter is opened and closed automatically. Under information, choose the name, concentration, operator.</li>
			<li>Under data, browse to desired folder. Click OK.</li>
		</ol>
		</li>
		<li>Acquire the spectra Measure&#62;Sample measurement, identify position(s) of cuvettes in sample changer 1-6 accordingly, click OK.</li>
	</ol>
	</li>
	<li>If taking a thermal denaturation transition:
	<ol>
		<li>Close Acquisition software File&#62;Exit and open a new program Variable Temperature Measurement&#62;Measure&#62;Parameters.</li>
		<li>Apply the following parameters to record a thermal transition, which can be adjusted as desired:
		<ol>
			<li>Under Temperature, select start temp: 4 &#176;C; interval: 0.2; target: 90 &#176;C; gradient: 1 &#176;C/min; wait 0 s. Under Start/End, select Start condition &#38; end condition as desired.</li>
			<li>Under General, select 3 Channels, CD/HT/Abs; bandwidth: 1 nm; wavelength: 270 nm (or as desired). Under Control, select none. Under Information, select name, concentration, operator.</li>
			<li>Under data, browse to desired folder. Click OK.</li>
		</ol>
		</li>
		<li>Cool samples to 4 &#176;C and wait 5 min at this temperature before obtaining a spectrum measurement.</li>
	</ol>
	</li>
	<li>Data work-up for spectra.
	<ol>
		<li>Using the function on the software, subtract the blank spectrum from the target acquisition to account for background signals arising from the buffer system in use: Spectra Analysis&#62;File&#62;Open.</li>
		<li>Once a blank file and a spectra file have been opened, drag View 2 under View 1 (on left hand side of screen).</li>
		<li>Click on Processing&#62;subtraction&#62;OK or exchange data&#62;OK.</li>
		<li>Extract data as an ASCII file: File&#62;Export and select ASCII.</li>
		<li>Use software to plot the corresponding spectrum. Smoothening of data is optional in cases where the signal-to-noise ratio is lower than expected. This transformation can be applied by applying the smoothening data option.</li>
	</ol>
	</li>
	<li>Data work-up for thermal denaturation transitions.
	<ol>
		<li>Extract data from the JASCO file as an ASCII file.</li>
		<li>Plot the ellipticity points as a function of temperature. Typically, smoothening of the data is necessary depending on the wavelength that is being examined. In some instances, the use of the wavelength at 210 nm displayed larger variations between plots that required smoothening. However, depending on the sample and concentrations, a number of calculations were carried out using the raw data.</li>
		<li>To calculate the thermal denaturation transitions (Tm) value, obtain a first derivative of the curve. The maxima or minima were an indication of this parameter. As in the previous step, some cases required smoothening of the data to obtain the most accurate value. 
		NOTE: Experiments were typically carried out in triplicate, which resulted in the corresponding average and standard deviation for the measurement.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The intent of this manuscript is to serve as a guide to researchers in the field, beginner or expert, to successfully achieve or enhance the synthesis of oligonucleotides of DNA or RNA. The described methodology focuses on the use of solid-phase synthesis using an automated DNA/RNA synthesizer via standard phosphoramidite chemistry. The report describes a step-by-step depiction of the synthesis, purification, and characterization of RNA dodecamers. In addition, the use of CD is employed to identify secondary structural m...

Solid-phase synthesis to obtain oligonucleotides of DNA/RNA is a powerful tool that has served several applications in various fields since the 1970s1,2,3 using phosphoramidite building blocks4. Examples of its broad influence include: its impact in labeling (via click chemistry reactions)5, structural probing6, and antisense technologies7, as well as its elucidation of biological mechanisms8,9, source as genetic material10, and the study of various natural and/or chemical modifications11,12, among many others.The modification that we use here represents the first step in our efforts to obtain RNA oligonucleotides that contain photoactive probes to enable temporal control of structure and function of this important biopolymer.
The synthesis of RNA dodecamers with sequences: 5&#39;-[CUA CGG AAU CAU]-3&#39;/5&#39;-[AUG AUU CCG UAG]-3&#39; (underlined positions represent the incorporation of a C2&#39;-O-thiophenylmethyl modification) constitutes the focus of this study. The sequences were chosen to enable the quantification and measurement of RNA strands as single strands, or as their corresponding duplex structures (no other secondary structures were predicted as thermodynamically stable). CD was used to establish the structural parameters, i.e., duplex formation and thermal denaturation transitions.
Synthesis 
The overall procedure for obtaining these oligonucleotides is illustrated in Figure 1 and follows the stepwise process: automated Solid-phase Synthesis &#8594; Deprotection &#8594; Purification &#8594; Quantification &#8594; Characterization. Figure 2 displays the monomeric units that are necessary in this procedure. The solid-phase synthesis of RNA is similar to that of DNA in that it is based on phosphoramidite chemistry (Figure 2, left) and the use of base-labile protecting groups for the nucleophilic exocyclic amines on G, A and C, e.g., acetyl, benzoyl, phenoxyacetyl, t-butyl or N,N-dimethylformamide (Figure 2, right). One more aspect to consider in RNA, due to the presence of the C2&#39;-OH group (lacking in the deoxyoligonucleotide biopolymers), is the additional step that has to be incorporated for the protection, and subsequent deprotection, of this nucleophilic position. In this respect, silicon-based protecting groups have become an attractive strategy due to their potential as biorthogonal moieties (specifically deprotected in the presence of fluoride), with the tert-butyldimethylsilyl (TBDMS) and triisopropylsilyloxymethyl (TOM) groups as popular choices (Figure 2, bottom-left).
In this work, the automated synthesis was carried out on a DNA/RNA synthesizer that uses standard phosphoramidite chemistry. The manufacturer settings on the instrument include an automated dilution step when using the commercial versions of the phosphoramidites for DNA, or the option to dilute at volumes set by the user. However, we decided to weigh the RNA phosphoramidite and dilute manually given that: 1) the price of the canonical phosphoramidites of RNA is higher (up to 50-times more expensive in some cases); 2) the modified phosphoramidites are often obtained in small amounts; and 3) the amount of wasted material upon using an automated dilution step (set by manufacturer) is large. In addition, we used: 1) commercially available solid supports (e.g., CPG) containing a protected nucleobase to function as the 3&#39;-end; and 2) commercial phosphoramidites (canonical nucleobases) protected with a TBDMS group at the C2&#39;-O-position. The detailed list of the synthesis steps is provided in Figure 3 and Table 1, along with further description and comments for steps that were adjusted for the RNA synthesis. Furthermore, Figure 4 illustrates the stepwise yields that are observed for every step after selecting the &#39;Trityl Monitor&#39; option, which quantitates the trityl cation released from each detritylation step.
It is worth noting that typically, in our experience, the limiting factor was obtaining the phosphoramidite containing the desired modification. That is, the development of a synthetic methodology that allows for the incorporation of modifications at select sites. In this report, we focus on the incorporation of a modified nucleotide for which we have established the corresponding synthetic methodology, the C2&#39;-O-thiophenylmethyl group. This group is small in size and does not affect the solid-phase synthesis in any manner. Since the incorporation of this group into oligonucleotides of RNA has been reported, along with structural and thermodynamic parameters4, no aspects of the organic synthesis leading to the modified phosphoramidites will be described herein.
Deprotection, Purification, and Characterization 
The deprotection of the exocyclic amines and &#223;-cyanoethyl groups occurs in the same step as that of the cleavage from the CPG-resin. We applied the commonly used conditions of heating the obtained resin in the presence of an aqueous solution of AMA, followed by cleavage of the C2&#39;-O-silyl groups in the presence of fluoride ions, and then purification via gel electrophoresis. While these have become standard conditions in many cases, modifications that are labile to basic conditions or fluoride ions may require milder conditions13,14, e.g., methanol/potassium carbonate (MeOH/K2CO3), or butylamine. Thus, a different set of protecting groups on the corresponding phosphoramidites is necessary. Furthermore, we chose electrophoresis as the preferred alternative to purify the deprotected oligomers given our previous experience with this method and the lack of other instrumentation. However, HPLC can alternatively be used as an effective method15. Characterization of the purified oligonucleotides was carried out via mass spectrometry, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), using a reported procedure by our group16.
Structural characterization and thermal stability of the obtained duplexes were carried out via CD. Specifically, we make use of CD to determine the thermal denaturation transitions of modified and unmodified oligonucleotides of RNA by following the decrease in ellipticity of the band at ca. 270 nm, as well as the disappearance of the band (with negative ellipticity) with a &#955;max at 210 nm. A spectra comparison before and after hybridization is provided to illustrate their differences and provide validation of the employed methodology. The use of CD is widely accepted in the determination of structural motifs in nucleic acids and aminoacids17, and can therefore be employed as a tool to determine various structural and thermodynamic parameters18; however, there are not many examples where the technique is used to assess thermal denaturation transitions. Some cases include the determination of thermal stabilities on DNA containing G-quadruplexes19,20 or in duplexes and hairpins of RNA21.
This report intends to provide the non-expert reader or viewer with a set of tools that enable a smooth start to this type of research. It will serve to enhance and compare with methodologies and techniques at other research laboratories that are involved in this exciting branch of science. The content in this report adds to the existing protocols of this technology from various sources, and enriches and facilitates the experience with a visual aid for each step.

<table><tbody><tr><td>AbsolveTM</td><td>PerkinElmer</td><td>6NE9711</td><td></td></tr><tr><td>Acetonitrile 99.9%, HPLC Grade</td><td>Fisher Scientific</td><td>75-05-8</td><td></td></tr><tr><td>Acetonitrile 99.9%, anhydrous for DNA sequencing</td><td>Fisher BioReagents</td><td>75-05-8</td><td></td></tr><tr><td>Acrylamide, 99+%</td><td>ACROS Organics</td><td>164850025</td><td></td></tr><tr><td>Ammonium chloride 98+%</td><td>Alfa Aesar</td><td>12125-02-9</td><td></td></tr><tr><td>Ammonium citrate, dibasic 98%</td><td>Sigma Aldrich</td><td>3012-65-5</td><td></td></tr><tr><td>Ammonium Fluoride&amp;nbsp; 98.0%, ACS grade</td><td>Alfa Aesar</td><td>12125-01-8</td><td></td></tr><tr><td>Ammonium hydroxide 28 - 30% in water, ACS Plus</td><td>Fisher Chemical</td><td>1336-21-6</td><td></td></tr><tr><td>Ammonium persulfate</td><td>ACROS Organics</td><td>1444</td><td></td></tr><tr><td>Argon-ultra high purity</td><td>&amp;nbsp;Airgas</td><td>7440-37-1</td><td></td></tr><tr><td>Bis-acrylamide Ultra pure</td><td>VWR-Amresco</td><td>172</td><td></td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>A73-1</td><td></td></tr><tr><td>Diethyl pyrocarbonate, 97%</td><td>ACROS Organics</td><td>A0368487</td><td></td></tr><tr><td>Ethanol, anhydrous, histological grade</td><td>Fisher Chemical</td><td>64-17-5</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt dehydrate 100.2%</td><td>Fisher Chemical</td><td>6381-92-6</td><td></td></tr><tr><td>Formamide&amp;nbsp;</td><td>Thermo Scientific</td><td>75-12-7</td><td></td></tr><tr><td>Hydrochloric acid, 36.5 - 38.0%, Certified ACS Plus</td><td>Fisher Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Magnesium chloride hexahydrate, 99%</td><td>Fisher Scientific</td><td>7786-30-3</td><td></td></tr><tr><td>Methanol, 99.9%,&amp;nbsp; HPLC Grade</td><td>Fisher Chemical</td><td>67-56-1</td><td></td></tr><tr><td>Methylamine 40% in water</td><td>Sigma Aldrich</td><td>74-89-5</td><td></td></tr><tr><td>1-Methyl-2-pyrrolidinone, andhydrous, 99.5%</td><td>Aldrich</td><td>872-50-4</td><td></td></tr><tr><td>Opti-TOFTM 96 Well Insert (123 x 81 mm)&amp;nbsp;</td><td>MDS SCIEX</td><td>1020157</td><td></td></tr><tr><td>RNase Away</td><td>Molecular BioProducts</td><td>7005-11</td><td></td></tr><tr><td>Sodium acetate, anhydrous 99.2%, Certified ACS</td><td>Fisher Chemical</td><td>127-09-3</td><td></td></tr><tr><td>Sodium chloride, 100.5%, Certified ACS</td><td>Fisher Chemical</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate 99.0%</td><td>Sigma</td><td>13472-35-0</td><td></td></tr><tr><td>2&amp;rsquo;,4&amp;rsquo; Triethylamine, 99+%</td><td>Alfa Aesar</td><td>121-44-8</td><td></td></tr><tr><td>TEMED</td><td>Amresco</td><td>761</td><td></td></tr><tr><td>Triethylamine trihydrofluoride, 98%</td><td>Aldrich</td><td>73602-61-6</td><td></td></tr><tr><td>Trifluoroacetic acid, 99%</td><td>Alfa Aesar</td><td>76-05-1</td><td></td></tr><tr><td>6&amp;rsquo;-Trihydroxyacetophenone monohydrate 98%</td><td>Sigma Aldrich</td><td>480-66-0</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP154-3</td><td></td></tr><tr><td>Urea</td><td>Fisher Scientific</td><td>U15-3</td><td></td></tr><tr><td>&lt;strong&gt;Reagents for the RNA synthesis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Deblocking mix, 3% trichloroacetic acid in dichloromethane</td><td>Glen Research</td><td>&amp;nbsp;40-4140-57</td><td></td></tr><tr><td>Cap Mix A, THF/Pyridine/Acetic anhydride</td><td>Glen Research</td><td>40-4110-52</td><td></td></tr><tr><td>Cap Mix B, 10% 1-methylimidazole in THF</td><td>Glen Research</td><td>40-4120-52</td><td></td></tr><tr><td>Activator, 0.25 M 5-ethylthio-1H-tetrazole in anhydrous acetonitrile</td><td>Glen Research</td><td>&amp;nbsp;30-3140-52</td><td></td></tr><tr><td>Oxidizing Solution, 0.02 M iodine in THF/Pyridine/Water</td><td>Glen Research</td><td>40-4330-52</td><td></td></tr><tr><td>U-RNA-CPG</td><td>Glen Research</td><td>20-3330-xx</td><td></td></tr><tr><td>Ac-G-RNA-CPG&amp;nbsp;</td><td>Glen Research</td><td>20-3324-xx</td><td></td></tr><tr><td>Ac-G-CE Phosphoramidite&amp;nbsp;</td><td>Glen Research</td><td>10-3025-xx</td><td></td></tr><tr><td>U-CE Phosphoramidite</td><td>Glen Research</td><td>10-3030-xx</td><td></td></tr><tr><td>Ac-C-CE Phosphoramidite</td><td>Glen Research</td><td>10-3015-xx</td><td></td></tr><tr><td>Bz-A-CE Phosphoramidite&amp;nbsp;</td><td>Glen Research</td><td>10-3003-xx</td><td></td></tr></tbody></table>

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.

The synthesis of RNA dodecamers containing zero, one, or two 2-thiophenylmethyl modifications at the C2&#39;-O-position is described along with its corresponding purification and characterization. Furthermore, a detailed description of the structural analysis that was carried out via CD is included.
The four strands of RNA (including a strand with a complementary sequence) were obtained via solid-phase synthesis, which ...

Watch this Scientific Journal Video about Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism at JoVE

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.

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 ...

protocol-for-solid-phase-synthesis-oligomers-rna-containing-2-o

Universidad San Sebastian

Research

JoVE Journal

Chemistry

18.9K Views.  University of Colorado Denver. This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2'-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

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

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 ...

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

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Developmental Biology

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

Biochemistry

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Biology

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 ...

Bioengineering

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

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RNA Secondary Structure Prediction Using High-throughput SHAPE