The authors thank Kerstin Glensk for preparing the MTases M.BseCI and Set7/9 and gratefully acknowledge funding by the Excellence Initiative of the German Federal and State Governments and RWTH Aachen University. The authors are happy to provide 6BAz and SeAdoYn or other cofactor analogues for collaborative research.

1. General Instructions
<ol>
	<li>Store aziridine cofactor 6BAz (in DMSO) and protein MTase Set7/9 at -80 &#176;C and all other reagents including double-activated cofactor SeAdoYn and DNA MTase M.BseCI (in 50% glycerol) at -20 &#176;C.</li>
	<li>Determine the concentration of 6BAz and SeAdoYn via UV/Vis spectroscopy using the extinction coefficients &#949;269nm&#160;(6BAz) = 16,000 cm-1 M-1 and &#949;260nm (SeAdoYn) = 15,400 cm-1 M-1 in deionized water. Determine the concentration of MTases by the Bradford assay or, if the extinction coefficient is available, via direct absorption at 280 nm.</li>
	<li>Try to avoid creating bubbles by intensive pipetting or vortexing to prevent loss of enzyme activity. Instead, mix by gently pipetting up and down.</li>
	<li>When adding aziridine cofactors from stock solutions in DMSO make sure that final DMSO concentration in the assay is less than 5%. Always include 10 mM magnesium ions in the assay buffer to prevent non-specific reactions with DNA.</li>
	<li>When adding double activated cofactors from acidic stock solutions use small volumes (highly concentrated stock solutions) to avoid pH changes and make sure that the pH of the assay solution does not change significantly. Avoid thiols, e.g., &#946;-mercaptoethanol or dithiothreitol (DTT), in the assay buffer because they can interfere with the click reaction by complexation of the required copper ions.</li>
</ol>
2. One-step Labeling of DNA via Aziridine Cofactors
<ol>
	<li>Sequence-specific Methyltransferase-Induced Label ing (SMILing) of plasmid DNA with M.BseCI DNA MTase and aziridine cofactor 6BAz.
	<ol>
		<li>Thaw the cofactor solution at 20 &#176;C and prepare the reaction mixtures on ice.</li>
		<li>In addition to the assay perform a &#8220;cofactor&#8221; control, to visualize any non specific modifications, and an &#8220; enzyme&#8221; control, to make sure that the MTase preparation is free of the natural cofactor AdoMet.</li>
		<li>For the assay mix 2 &#181;l of 10x modification buffer (containing 100 mM Tris-HCl, 100 mM MgCl2, 20 mM &#946;-mercaptoethanol, pH 7.4), 2 &#181;l of pBR322 (0.5 &#181;g/&#181;l), 10 eq. M.BseCI per recognition sequence on the DNA (1 recognition sequence in pBR322) and the aziridine cofactor 6BAz to a final concentration of 60 &#181;M within a total volume of 20 &#181;l. Add cofactor and DNA MTase last. 
		NOTE: &#946;-Mercaptoethanol is toxic, corrosive and environmentally damaging.</li>
		<li>For the &#8220;cofactor&#8221; control add deionized water instead of M.BseCI and for the &#8220;enzyme&#8221; control add deionized water instead of 6BAz.</li>
		<li>Mix the solutions by gently pipetting up and down.</li>
		<li>Incubate the tubes at 55 &#176;C for 1 hr.</li>
		<li>Centrifuge briefly to collect all liquid at the bottom of the tubes.</li>
	</ol>
	</li>
	<li>Restriction-modification assay to verify DNA modification.
	<ol>
		<li>Prepare a solution by mixing 10 &#181;l 10x R.TaqI buffer (containing 100 mM Tris-HCl, 50 mM MgCl2, 1 M NaCl, 1 mg/ml bovine serum albumin, pH 8.0), 80 &#181;l deionized water and 3.3 &#181;l of the restriction endonuclease (REase) from Thermus aquaticus (R.TaqI, 10 U/&#181;l). Make sure to add the REase in the last step.</li>
		<li>To each tube from 2.1.7 add 2 &#181;l of 10x R.&#160;TaqI buffer and 28 &#181;l of the solution from above (2.2.1).</li>
		<li>Mix the solutions by gently pipetting up and down.</li>
		<li>Incubate the tubes at 65 &#176;C for 30 min.</li>
		<li>Centrifuge briefly to collect all liquid at the bottom of the tubes.</li>
	</ol>
	</li>
	<li>Electromobility shift assay (EMSA) with streptavidin to verify functional modification.
	<ol>
		<li>Remove 25 &#181;l from each tube (2.2.5) and add 2.4 &#181;l of a streptavidin solution (1 mM with respect to streptavidin monomer in streptavidin buffer containing 100 mM Na2HPO4, 100 mM NaCl, pH 7.5; 4 equivalents of total biotin). Add 2.4 &#181;l of streptavidin buffer to the remaining tubes.</li>
		<li>Incubate all tubes at 37 &#176;C for 1 hr.</li>
	</ol>
	</li>
	<li>Analysis via agarose gel electrophoresis.
	<ol>
		<li>Add 5 &#181;l of 6x loading buffer (0.25% bromophenol blue, 30% glycerol) to each tube.</li>
		<li>Mix the solutions gently.</li>
		<li>Load 10 &#181;l of each sample into the wells of an agarose gel (1% agarose in 0.5x TBE buffer containing 1x GelRed from a 10,000x stock solution).</li>
		<li>Run the gel in 0.5x TBE buffer with 80 V for approx. 1 hr.</li>
		<li>Visualize DNA bands on a UV table (312 nm) with a CCD camera equipped with a filter (540 &#177; 50 nm). 
		NOTE: UV light is damaging to eyes and skin.</li>
	</ol>
	</li>
</ol>
3. Two-step Protein Labeling via Double Activated Cofactors
<ol>
	<li>Methyltransferase-Directed Transfer of Activated Groups (mTAG) with Set7/9 and double-activated cofactor SeAdoYn for histone H3 lysine 4 labeling (modification step).
	<ol>
		<li>Thaw the components and prepare the reaction mixtures on ice. NOTE: Always keep SeAdoYn cooled to avoid degradation.</li>
		<li>In addition to the assay perform a &#8220;cofactor&#8221; control, to visualize any non specific modifications, and an &#8220;enzyme&#8221; control, to exclude non-specific reactions of the fluorescent probe.</li>
		<li>Prepare an assay solution (20 &#181;l) containing modification buffer (50 mM Tris-HCl, 5% glycerol, pH 8.5), 10 &#181;M histone H3, 10 &#181;M Set7/9 and 600 &#181;M SeAdoYn (mixture of both epimers at selenium). In the last steps add cofactor and then MTase.</li>
		<li>For the &#8220;cofactor&#8221; control prepare an assay solution as in 3.1.3 and add 60 mM AdoMet to compete with the synthetic cofactor. For the &#8220;enzyme&#8221; control add deionized water instead of SeAdoYn.</li>
		<li>Mix the solutions by slowly pipetting up and down. Check the pH by adding 1 &#181;l of each solution to the upper field of a pH strip (pH range 5 - 10).</li>
		<li>Incubate at 37 &#176;C for 2 hr.</li>
		<li>In the meanwhile prepare a 12% SDS polyacrylamide gel (running gel: 357 mM Bis-Tris pH 6.5-6.8, 0.1% (w/v) APS, 0.04% (v/v) TEMED and 12% acrylamide/bisacrylamide 37.5:1; loading gel: 357 mM Bis-Tris pH 6.5-6.8, 0.1% (w/v) APS, 0.04% (v/v) TEMED and 5% acrylamide/bisacrylamide 37.5:1). 
		NOTE: Acrylamide/bisacrylamide is toxic and health hazardous. Wear gloves during this procedure.</li>
	</ol>
	</li>
	<li>Chemical labeling of alkinylated lysine 4 in histone H3 via copper-catalyzed azide-alkyne cycloaddition (CuAAC) (labeling step).
	<ol>
		<li>Just before the end of the modification reaction prepare a 5x click mix containing 3 mM CuSO4, 3 mM tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA), 250 mM sodium ascorbate and 6 mM TAMRA azide with a total volume of 20 &#181;l.</li>
		<li>Add 5 &#181;l of the freshly prepared 5x click mix to each tube to start the CuAAC and quench the modification reaction.</li>
		<li>Mix gently by pipetting up and down.</li>
		<li>Protect all tubes with aluminum foil from light to avoid photo-bleaching of the fluorophore.</li>
		<li>Incubate at 20 &#176;C for 1 hr.</li>
	</ol>
	</li>
	<li>Protein precipitation to remove excess of free TAMRA fluorophore.
	<ol>
		<li>To avoid outshining of the fluorescent labeled histone H3 by intensive in-gel fluorescence of free TAMRA fluorophore, remove excess fluorophore by precipitation of proteins (3.3.2 &#8211; 3.3.4)34.</li>
		<li>Add 75 &#181;l methanol, 18.8 &#181;l chloroform and 50 &#181;l deionized water to each tube and vortex briefly after each addition. Centrifuge at 16,000 x g for 5 min. Remove the upper phase without disturbing the interface layer, which contains the protein.</li>
		<li>Add 56.3 &#181;l methanol to the remaining phase in each tube, vortex and centrifuge at 16,000 x g for 5 min to pellet the protein. Remove the supernatant. Repeat this step to wash the pellet.</li>
		<li>Cover the open tubes with a lint free tissue and let them dry for 15 &#8211; 30 min.</li>
	</ol>
	</li>
	<li>Analysis via SDS PAGE.
	<ol>
		<li>Dissolve the precipitated proteins from 3.3.4 in 20 &#181;l SDS loading buffer (50 mM Tris-HCl, 2.5% (w/v) SDS, 10% (v/v) glycerol, 320 mM &#946;-mercaptoethanol and 0.05% (w/v) bromophenol blue, pH 6.8). Make sure to completely dissolve the pellet by rinsing the walls of the tubes with a pipette.</li>
		<li>Incubate the samples at 95 &#176;C for 10 min and let them cool down to 20 &#176;C.</li>
		<li>Centrifuge briefly to collect all liquid at the bottom of the tubes.</li>
		<li>Load the whole amount of each sample into the wells of an SDS polyacrylamide gel (3.1.7). Use 50 mM MOPS, 50 mM Tris-X (Tris-base), 5 mM EDTA, 0.1% (w/v) SDS as running buffer for electrophoresis.</li>
		<li>Run the gel with 120 V for approx. 90 min.</li>
		<li>Visualize the in-gel fluorescence on a UV table (312 nm) with a CCD camera equipped with a filter (540 nm &#177; 50 nm). 
		NOTE: UV light is damaging to eyes and skin.</li>
	</ol>
	</li>
</ol>

The authors disclose the following competing financial interest: E.W. is inventor on related patents.

One-step labeling of DNA with DNA MTases and aziridine cofactors (SMILing DNA) is a robust method but some aspects should be considered when planning the experiment.
Aziridine cofactor: The 6BAz concentration for DNA labeling with M.BseCI was 60 &#181;M. When using other DNA MTases the cofactor concentration should be optimized, e.g. concentrations as low as 20 &#181;M have been employed with the DNA MTase M.TaqI19. Low 6BAz concentrations have the advantage that a...

Specific labeling of nucleic acids1,2 and proteins3,4 is of major interest for functional characterizations, medical diagnosis and (nano)biotechnology. Here we present an enzymatic labeling method for these biopolymers which is based on S-adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTases). This class of enzymes (EC 2.1.1.) targets individual nucleophilic positions (nitrogen, oxygen, sulfur and carbon atoms) within specific residues of nucleic acids and proteins and naturally transfers the activated methyl group of the cofactor AdoMet (Figure 1A)5. In addition, MTases can utilize synthetic cofactor analogues for specific labeling with affinity tags, fluorophores or other labels (Figure 1B)6. Two classes of AdoMet analogues have been developed: Aziridine cofactors for Sequence-specific Methyltransferase-Induced Labeling (SMILing)7 and double activated AdoMet analogues for methyltransferase-directed Transfer of Activated Groups (mTAG)8.
<img alt="Figure 1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> 
Figure 1: Reactions catalyzed by methyltransferases (MTases). A. Methyl group transfer from the natural cofactor AdoMet (SAM) to various substrates including DNA, RNA, proteins and small biomolecules. B. Labeling/functionalization of nucleic acids and proteins (NNNNN = base pairs for DNA, nucleotides for RNA and amino acids for proteins; XXXXX = recognition sequence of the MTase with target residue in green) with synthetic cofactor analogues. Aziridine cofactors containing a reporter group (blue sphere) attached to the adenine ring are sequence specifically coupled with the target residue (left) and double-activated AdoMet analogues lead to transfer of extended alkyl chains carrying a chemical reporter Y (right) which can be labeled by bioorthogonal click reaction in a second step. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Aziridine cofactors work best with DNA MTases. They contain a three membered ring with a nitrogen atom9 (or an N-mustard10,11) instead of the sulfonium center as reactive group. Protonation of this nitrogen atom activates the aziridine ring for nucleophilic attack by the target nucleotide which leads to covalent coupling of the whole cofactor with DNA. By attaching reporter groups to the adenine ring the aziridine cofactors can be used in combination with DNA MTases to label DNA in one step (Figure 1B, left)7,12. This is demonstrated in detail for the biotinylation of DNA with 6BAz13&#8211;15 (aziridine cofactor with biotin attached to the 6 position of the adenine ring) and the adenine-specific DNA MTase from Bacillus stearothermophilus (M.BseCI)16 (Figure 2, see protocol section 2: One-step labeling of DNA via aziridine cofactors). In addition to M.BseCI (5&#8217;-ATCGAT-3&#8217; recognition sequence), the DNA MTases from Thermus aquaticus (M.TaqI, 5&#8217;-TCGA-3&#8217;), from Haemophilus heamolyticus (M.HhaI, 5&#8217;-GCGC-3&#8217;) and from Spiroplasma (M.SssI, 5&#8217;-CG-3&#8217;) have been successfully used to biotinylate DNA with 6BAz17. Furthermore, aziridine cofactors can be employed for one-step fluorescence DNA labeling18,19.
<img alt="Figure 2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> 
Figure 2: Sequence specific one-step biotinylation of DNA with M.BseCI and 6BAz. The DNA MTase M.BseCI recognizes the double-stranded DNA sequence 5&#8217;-ATCGAT-3&#8217; and naturally methylates the amino group of the second adenine residue (green) using AdoMet. With the aziridine cofactor 6BAz the course of the reaction is changed and M.BseCI leads to sequence specific DNA biotinylation by coupling the whole cofactor including biotin (blue) with the target adenine. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Double activated AdoMet analogues contain extended unsaturated side chains instead of a methyl group at the sulfonium center (Figure 1B, right)20. The unsaturated double or triple bond in &#946;-position to the sulfonium center electronically compensates unfavorable steric effects within the transition state by conjugative stabilization. Since both the sulfonium center and the unsaturated bond activate the side chain for enzymatic transfer, these cofactors were named double-activated AdoMet analogues. Typically, they are used to transfer side chains with unique chemical groups (chemical reporters), like amino, alkyne and azide groups, for chemo-selective labeling in a second step8,21. In general, double-activated AdoMet analogues can not only function as cofactors for DNA MTases8,20,21 but also for RNA MTases22,23 and protein MTases24&#8211;28 allowing additional labeling of RNA and proteins. However, the extended side chains are sterically more demanding than a methyl group and enlarging the MTase active sites by protein engineering is often required to obtain efficient transfer rates. Another solution to this problem is to use an AdoMet analogue with a small propargyl group (three carbons) where the terminal alkyne serves two functions: 1. Stabilization of the transition state during enzymatic transfer and 2. reactive handle for following chemical modifications by copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. It turned out that the resulting propargylic AdoMet analogue29 is quite unstable under neutral or slightly basic conditions and only of limited use. This drawback can be fixed by replacing the sulfur atom with selenium. The resulting cofactor 5&#8216;-[(Se)[(3S)-3-amino-3-carboxypropyl]prop-2-ynylselenonio]-5&#8216;-deoxyadenosine (SeAdoYn, Figure 3) is accepted by wild-type DNA, RNA and protein MTases30&#8211;32 which abrogate the need for protein engineering in many cases. This is exemplified by fluorescence protein labeling with the histone H3 lysine 4 (H3K4) MTase Set7/933 (Figure 3, see protocol section 3: Two-step protein labeling via double activated cofactors).
<img alt="Figure 3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> 
Figure 3: Sequence-specific two-step fluorescence labeling of histone H3 with Set7/9, SeAdoYn and TAMRA azide. The protein MTase Set7/9 naturally methylates the amino group of lysine 4 in histone H3 (H3K4, green) using AdoMet. With the double-activated cofactor SeAdoYn the MTase transfers a small propargyl group (red) to the lysine residue. The attached terminal triple bond is then selectively modified in a bioorthogonal click reaction (copper-catalyzed azide-alkyne cycloaddition, CuAAC) with azide-derivatized TAMRA (tetramethylrhodamine, blue) fluorophore. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Reagent/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">6BAz</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Weinhold et al., Patent number US 8,129,106, published March 6, 2012.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">&#946;-Mercaptoethanol</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">28625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acetic acid</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10304980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">10688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">UltraPure Agarose</td>
			<td style="width:139px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ammonium persulfate (APS)</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">13375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bis-Tris</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Boric acid</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bromophenol blue Na salt</td>
			<td style="width:139px;">Serva</td>
			<td align="right">15375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Copper(II) sulfate</td>
			<td style="width:139px;">Aldrich</td>
			<td style="width:119px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Chloroform</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right">10020090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Coomassie Brilliant Blue</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">17525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EDTA disodium salt</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">GelRed (10000x in water)</td>
			<td style="width:139px;">Biotium</td>
			<td align="right" style="width:119px;">41003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Glycerol (99.5%)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">FastRuler Low Range DNA Ladder</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM1103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Histone H3</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Philipp Voigt and Prof. Danny Reinberg; expression and isolation according to T. J. Richmond et al., J. Mol. Biol. 1997, 272, 301-311.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M.BseCI</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Michael Kokkinidis; expression and isolation according to Kapetaniou et al., Acta Cryst. 2006, F63, 12-14.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Methanol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10675112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesiumchloride&#160; hexahydrate</td>
			<td style="width:139px;">J.T. Baker</td>
			<td align="right" style="width:119px;">4003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MOPS</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium chloride</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pH strip (Neutralit)</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">1,095,330,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pBR322</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SD0041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">R.TaqI (10u/&#181;l)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>ER0671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SeAdoYn</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Willnow et al., ChemBioChem 2012, 13, 1167-1173.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Set7/9</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Prof. Danny Reinberg, expression and isolation according to D. Reinberg et al., Genes Dev.2002, 16, 479-489.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Streptavidin</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">(+)-Sodium L-ascorbate</td>
			<td style="width:139px;">Sigma Life Science</td>
			<td style="width:119px;">A7631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SDS Granular</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">di-Sodiumhydrogenphosphat</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">106,586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TAMRA-azide</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TaqI buffer (10x)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">B28</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:245px;">N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td style="width:139px;">Acros Organics</td>
			<td align="right" style="width:119px;">42058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-HCl</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-X (TRIS-base)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">762342</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

One-step Labeling of DNA via Aziridine Cofactors
This example reaction is carried out with the DNA MTase M.BseCI, which modifies the second adenine residue within the double-stranded 5&#8217;-ATCGAT-3&#8217; sequence and has one recognition site on the pBR322 plasmid (Figure 4A). To test plasmid labeling, pBR322 is challenged with the restriction endonuclease (REase) R.TaqI (5&#8216;-TCGA-3&#8216;). R.TaqI has seven sites on pBR322, one of which is in...

Watch this Scientific Journal Video about Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues at JoVE.com

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

sequence-specific-labeling-nucleic-acids-proteins-with

Research

JoVE Journal

Biology

13.9K Views.  RWTH Aachen University. DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Video: Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.