Name: protocol for the solid-phase synthesis of oligomers of rna containing a 2'-o-thiophenylmethyl modification and characterization via circular dichroism
Uploaded: 2017-07-28T15:00:00
Description: 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

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 Amidit...

<ol>
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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.

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			<td height="20" style="height:20px;width:445px;">AbsolveTM</td>
			<td style="width:152px;">PerkinElmer</td>
			<td style="width:117px;">6NE9711</td>
			<td style="width:64px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, HPLC Grade</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, anhydrous for DNA sequencing</td>
			<td>Fisher BioReagents</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide, 99+%</td>
			<td>ACROS Organics</td>
			<td>164850025</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium chloride 98+%</td>
			<td>Alfa Aesar</td>
			<td>12125-02-9</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium citrate, dibasic 98%</td>
			<td>Sigma Aldrich</td>
			<td>3012-65-5</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium Fluoride&#160; 98.0%, ACS grade</td>
			<td>Alfa Aesar</td>
			<td>12125-01-8</td>
			<td></td>
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			<td height="20" style="height:20px;">Ammonium hydroxide 28-30% in water, ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium persulfate</td>
			<td>ACROS Organics</td>
			<td>1444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Argon-ultra high purity</td>
			<td>&#160;Airgas</td>
			<td>7440-37-1</td>
			<td></td>
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			<td height="20" style="height:20px;">Bis-acrylamide Ultra pure</td>
			<td>VWR-Amresco</td>
			<td>172</td>
			<td></td>
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			<td height="20" style="height:20px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A73-1</td>
			<td></td>
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			<td height="20" style="height:20px;">Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
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			<td height="20" style="height:20px;">Ethanol, anhydrous, histological grade</td>
			<td>Fisher Chemical</td>
			<td>64-17-5</td>
			<td></td>
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			<td height="20" style="height:20px;">Ethylenediamine tetraacetic acid, disodium salt dehydrate 100.2%</td>
			<td>Fisher Chemical</td>
			<td>6381-92-6</td>
			<td></td>
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			<td height="20" style="height:20px;">Formamide&#160;</td>
			<td>Thermo Scientific</td>
			<td>75-12-7</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid, 36.5-38.0%, Certified ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>7647-01-0</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate, 99%</td>
			<td>Fisher Scientific</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol, 99.9%,&#160; HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methylamine 40% in water</td>
			<td>Sigma Aldrich</td>
			<td>74-89-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Methyl-2-pyrrolidinone, andhydrous, 99.5%</td>
			<td>Aldrich</td>
			<td>872-50-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-TOFTM 96 Well Insert (123 x 81 mm)&#160;</td>
			<td>MDS SCIEX</td>
			<td>1020157</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase Away</td>
			<td>Molecular BioProducts</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium acetate, anhydrous 99.2%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>127-09-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride, 100.5%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic dihydrate 99.0%</td>
			<td>Sigma</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2&#8217;,4&#8217; Triethylamine, 99+%</td>
			<td>Alfa Aesar</td>
			<td>121-44-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>Amresco</td>
			<td>761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triethylamine trihydrofluoride, 98%</td>
			<td>Aldrich</td>
			<td>73602-61-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid, 99%</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6&#8217;-Trihydroxyacetophenone monohydrate 98%</td>
			<td>Sigma Aldrich</td>
			<td>480-66-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP154-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urea</td>
			<td>Fisher Scientific</td>
			<td>U15-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents for the RNA synthesis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deblocking mix, 3% trichloroacetic acid in dichloromethane</td>
			<td>Glen Research</td>
			<td>&#160;40-4140-57</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix A, THF/Pyridine/Acetic anhydride</td>
			<td>Glen Research</td>
			<td>40-4110-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix B, 10% 1-methylimidazole in THF</td>
			<td>Glen Research</td>
			<td>40-4120-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activator, 0.25 M 5-ethylthio-1H-tetrazole in anhydrous acetonitrile</td>
			<td>Glen Research</td>
			<td>&#160;30-3140-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxidizing Solution, 0.02 M iodine in THF/Pyridine/Water</td>
			<td>Glen Research</td>
			<td>40-4330-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-RNA-CPG</td>
			<td>Glen Research</td>
			<td>20-3330-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-RNA-CPG&#160;</td>
			<td>Glen Research</td>
			<td>20-3324-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-CE Phosphoramidite&#160;</td>
			<td>Glen Research</td>
			<td>10-3025-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3030-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-C-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3015-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bz-A-CE Phosphoramidite&#160;</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 ...

The overall goal of this report is to illustrate the procedure to obtain oligonucleotides of RNA via solid-phase synthesis. Dodecamers of RNA will be synthesized as models and their purification and characterization will also be demonstrated. Through its visual component, this publication will broaden the scope of the methodologies and facilities used to researchers in various fields.Providing a visual aid, along with a little protocol will enable researchers to employ these techniques. One advantage of this technique is its amenability to a wide range of modifications. As today's applications become more demanding, it is important to have a standard protocol.Phosphoramidite chemistry is experiencing a resurgence due to the robustness of the method and its potential for obtaining a oligonucleotides for therapeutic applications. While purchasing oligonucleotides is attractive, price and availability of modifications shift interest towards preparing these oligomers in-house, thus making this procedure of utmost importance. To begin this procedure, weigh each phosphoramidite into oven-dried 10-milliliter amber bottles and cap each bottle with a septum previously perforated with a 20-gauge needle.Place the bottles immediately under vacuum using a dessicator containing a drying agent. After filling the dessicator with dry argon, remove each bottle and immediately add anhydrous acetonitrile before securing onto a solid-phased peptide synthesizer. Next, remove the septa from each bottle, and place on the synthesizer via a bottle-change function.To set up the solid-phase synthesis, select the software icon. Select the synthesizer name, and click on OK.Then, create a new synthesis by selecting File, and New Synthesis Order. Following this, use the prompt window to enter the run date, customer, and run ID.Select the instrument, sequence name, and sequence.Under the Cycle tab, assign the previously created method. Then, choose the End Procedure manual, which leaves the oligonucleotide bound to the CPG resin. Select DMT Off.Save the file by selecting File and Save As.Then, provide a name for the experiment and click on Save. Next, send the file to begin synthesis by selecting Order and Send Order to Synthesizer. On the prompt window, select a column position and click on OK.On the Synthesizer window, select Trityl Monitor.Click on Choose Function, and Trityl Monitor every step by typing 1. Begin the synthesis by selecting Synthesizer and Prepare to Start. Place the columns with the desired three-prime end on the positions indicated on the instrument.Then, click Start and No on the instrument. Once the synthesis is complete, remove the column from the instrument and place it in a round-bottomed flask. Dry the column under reduced pressure for about 0.5 hours.Transfer the white resin from the column to a 1.6-milliliter centrifuge tube, and add 0.5 milliliters of an aqueous methylamine and ammonia solution. Secure the centrifuge tube cap with parafilm, and heat the contents to 60 degrees Celsius for one and a half hours. Place a heavy object on top of the tubes to ensure the concentration is kept constant.After cooling to room temperature, briefly centrifuge the sample to spin down contents. Then, transfer the supernatant to a new centrifuge tube. Cool the tube to 70 degrees Celsius by placing in a dry-ice ethanol bath for five minutes, and then concentrate until dry using a speed vac concentrator.Now, resuspend the solids in 0.4 milliliters of an amine trihydrafluoride solution, and then secure the cap with laboratory film. Heat the solution to 60 degrees Celsius for one and a half hours. Place a heavy object on top of the tubes to ensure constant concentration.After cooling the solution to room temperature, add 0.04 milliliters of a sodium acetate solution followed by gentle mixing with a pipette tip. Add one milliliter of ethanol and cool the solution to approximately 70 degrees Celsius with a dry-ice ethanol bath for 15 minutes. Following this, centrifuge the solution at 15, 000 RPM at four degrees Celsius for ten minutes.Use a pipette to extract the supernatant and dry the resulting pellet under reduced pressure. After drying, resuspend the obtained solid and loading buffer, and mix until the solution is homogenous. Then, load the suspension onto a previously prepared polyacrylamide gel.Apply a current through the gel until the bromethymol blue marker is located between one-half to two-thirds down the gel. After removing the gel from the stand, transfer the gel contents from the glass to the plastic wrap and place over a thin layer chromatography plate covered with silica, to visualize the bands using a UV lamp. Use a marker to delineate the position where the upper band is located, and cut it using a razor blade.Place the gel containing the RNA into a 50-milliliter conical tube, and crush it to small pieces using a glass rod. Now, suspend the gel residues into an aqueous sodium chloride solution, and shake the suspension at 37 degrees Celsius for 12 hours. Transfer the suspension into a 15-milliliter tube, and then centrifuge at 3400 RPM for ten minutes.To desalt the sample using a reverse-phase C18 cartridge, first wash the cartridge with acetonitrile, water, aqueous ammonium chloride, and the RNA solution, leaving the gel residues behind. After washing with water, elute the RNA from the column using three milliliters of 60%aqueous methanol solution into three different test tubes. Cool the tubes to 70 degrees Celsius in a dry ice bath for five minutes, and then concentrate the contents to dryness using a speed vac concentrator.After concentrating the eluant under reduced pressure, redissolve the residue in 0.3 milliliters of RNase-free water. After diluting the solution, deposit one microliter on the UV vis instrument to measure the UV vis spectrum between 200 and 450 nanometers. For MALDI-TOF analysis, first wash a C18 pipette tip with 50%acetonitrile.Equilibrate the pipette tip with 0.1%trifluoroacetic acid. Then, load the pipette tip with a solution containing 100 to 150 picomoles of sample. After washing the pipette tip with 0.1%TFA and water, elute the sample into a solution containing the desired matrix.Deposit 0.9 microliters of the solution onto a MALDI plate and allow the sample to dry. For RNA structure analysis, transfer the sample containing RNA into a microcuvette and position in the sample changer of a circular dichroism instrument. Open the nitrogen tank to provide a flow that moves the air floater located on the instrument to approximately 40.Then, turn on the cooler. Now, turn on the instrument. Open the software by clicking on the Spectrum Manager icon, and then open the Acquisition window by clicking on Spectrum Measurement.After purging the instrument with nitrogen, acquire the circular dichroism spectra by selecting Measure and Parameters. Under the General tab, select 200-350 nanometers for the scan, CD and HT for the channels, 0.1 nanometers for the data pitch, 100 nanometers per minute for the scanning speed, one nanometer for the bandwidth, and five for the number of accumulations. Under the Cell Unit tab, chose 20 degrees Celsius.Under the Control tab, select Shutter is opened and closed automatically. Under the Information tab, input the sample name, operator, concentration, and solvent. Under the Data tab, browse to desired folder, and click on OK.Acquire the spectra by clicking on Measure, and Sample Measurement.Finally, identify the positions of the cuvettes in sample changer one through six, and click on OK.The four strands of RNA were obtained via solid-phase synthesis, and following purification yielded between 300 and 700 nanomoles of each oligonucleotide. The MALDI-TOF chromatograms of the oligonucleotides are shown here. No major difference in the normalized UV vis spectra is observed from comparison of the band at 260 nanometers and canonical and modified oligonucleotides, whether comparing single-stranded samples or duplex structures.However, minor changes are observed upon measurement of their circular dichroism spectra. In addition, Tm measurements of the three duplex structures displayed a distinct value that was indicative of the destabilization induced by the incorporation of the two prime othyo phenylmethyl modification on one of the strands. It is important to remember that the yield of the solid-phase synthesis is depending on reagent purity.Moisture will have a negative impact on each step, cause the amount of water present must be reagent at all time. The reported methodology is meant to serve as a general resource for scientists. The technique may vary depending on the desired modification, available instrumentation, or time constraints.The type of chemistry employed herein represents a very robust methodology that has been in use since the 1980s, and is still heavily used in academic as well as industrial settings. After watching this video, you should be able to perform the synthesis, purification and characterization of your own modified or canonical oligonucleotides of RNA or DNA. Don't forget that working with the necessary reagents can be hazardous, and protective equipment such a safety goggles, gloves, and lab coats should be worn at all times.All reagents should be handled with care, and inside a fume hood when appropriate.

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