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.

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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>
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		<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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		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride 98+%</td>
			<td>Alfa Aesar</td>
			<td>12125-02-9</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>
		</tr>
		<tr height="20">
			<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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		<tr height="20">
			<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Argon-ultra high purity</td>
			<td>&#160;Airgas</td>
			<td>7440-37-1</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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous, histological grade</td>
			<td>Fisher Chemical</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<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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		<tr height="20">
			<td height="20" style="height:20px;">Formamide&#160;</td>
			<td>Thermo Scientific</td>
			<td>75-12-7</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid, 99%</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
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		<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>
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			<td height="20" style="height:20px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP154-3</td>
			<td></td>
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			<td height="20" style="height:20px;">Urea</td>
			<td>Fisher Scientific</td>
			<td>U15-3</td>
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			<td height="20" style="height:20px;">Reagents for the RNA synthesis:</td>
			<td></td>
			<td></td>
			<td></td>
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		<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 ...

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

Universidad de Cartagena UDEC

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

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.

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

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Chapters in this video

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

Synthesis and Characterization of Functionalized Metal-organic Frameworks

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

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphos^Ph Complexes

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

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Synthesis and Characterization of Supramolecular Colloids

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

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