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.

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

Research

JoVE Journal

Chemistry

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 ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.