This protocol outlines how you can produce RNA functionalized with alkynes for labeling with either fluorophores, biotin or other things of interest for detection or crosslinking.

🚛 Reagents needed 📦 :

Replace the natural base by only 1-15% with the alkyne modified one since modified bases are not as well tolerated by polymerases as natural ones. Optimize depending on your yield. Data presented are for 1.8 Kb amplification with 1 µg dsDNA template. Longer transcripts are preferentially amplified.

Protocol

Start an In Vitro Transcription reaction on ice by mixing the following:

Incubate for 24h ⏰🛌 at 37°C.

Add 30 µl of nuclease-free water to bring the reaction to 50 µl 

 Add 100 µl Oligo Binding Buffer to 50 µl sample.

 Add 400 µl ethanol (95-100%) and mix well by pipetting.

 Transfer the 450 µl sample to the Zymo spin column.

 Add 750 µl DNA Wash Buffer to the column and centrifuge for 1 min at 10,000-16,000 x g.

 Transfer the column to a new Eppendorf tube.

 Add 7 µl elution buffer (water or TE) to the center of the column.

 Centrifuge for 1 min at 10,000-16,000 x g.

The flow-through can immediately be used or stored frozen.
Measure the yield on NanoDrop.

Pause point. Store in -20°C until you want to label the RNA with your functional group.

Labeling using CuAAC:

🚛 Reagents needed 📦 :

✋ Notes 🛑❗

Ascorbic acid is very unstable in aqueous solution because of its oxidizing behavior. In water ascorbic acid oxidises easily to dehydroascorbic acid, which has an orange-brown color. Oxygen and light exposure will speed up this oxidation reaction, this reaction is reversible, but sodium ascorbate is needed for the conversion of Cu(II) to Cu(I), and Cu(I) is needed for the copper click conjugation reaction. If you see the reaction turning brownish or yellow you can be certain that the reaction failed and that  you have probably damaged the oligo through Cu(II) oxidative DNA damage.

Because of this it is important to avoid oxygen in the reaction. Never work with water that has been stored in small containers for long time such as Eppendorf tubes. Take water from larger vessels. The argon wine preserver is used to degas all liquids and the reaction before sealing the reaction tube for incubation. Because dehydroascorbic acid easily decomposes further and this reaction is not reversible it is important to work quickly from the time that (+)-Sodium L-ascorbate is dissolved in water (start the reaction within 5 min or less).

Lastly mix all the reagents fresh just before you are going to use them.

1️⃣ Use an analytical balance and weight up the following chemicals: 

Add the CuSO4 in a 15 ml Falcon tube, the sodium ascorbate in an Eppendorf tube and the BTTAA also in an Eppendorf tube. 

Measure a known amount of the stock oligo on the NanoDrop (this will be used to calibrate and measure the final yield in % later).

2️⃣ 20 mM CuSO4 stock.

Add 2.3 mL of nuclease-free water to the 11.6 mg CuSO4 to get a 20 mM stock. It will take some time for the CuSO4 to fully dissolve. Inspect the tube and make sure it is fully dissolved.

3️⃣ 100 mM BTTAA stock.

The BTTAA easily sticks to the side of the tube so spinndown with a benchtop centrifuge.

Add 100 µl of nuclease-free water to the 4.3 mg of BTTAA to get a 100 mM stock.

4️⃣ Mix together BTTAA and CuSO4

Make the following master mix by adding the 100 mM BTTAA to the 20 mM CuSO4.

The reaction will immediately turn blue 🔵.
Optional: degas the reaction with argon and let it sit on the table while you are preparing the rest of the reaction.

5️⃣ Mix oligonucleotide and azide together.

Take 1.8 µl of the 10 mM azide stock and dilute with 16.2 µl by adding nuclease-free water to get 1 mM.
Ad 6 µl of the amplified RNA (aRNA) to the reaction.

6️⃣ 300 mM sodium ascorbate

Add 1 mL of nuclease-free water to the 60 mg of sodium ascorbate to get a 300 mM stock. Do not vortex but rather flick the tube and turn it upside down a couple of times until the flakes have fully dissolved. Once dissolved work quickly to start the reaction.

7️⃣ Mix BTTAA, CuSO4 and sodium ascorbate to a master mix

As soon as the sodium ascorbate is fully dissolved add to the 200 µl [CuSO4 + BTTAA] mix 100 µl of 300 mM sodium ascorbate.

The reaction will immediately turn back to transparent from the [CuSO4 + BTTAA] blue color  🔵��➡️ ⚪.

Degas the [CuSO4 + BTTAA + sodium ascorbate] reaction by putting down the argon can nozzle into the mixture and carefully press down the valve of the can so the mixture is bubbling for a couple of seconds.

8️⃣ Start the reaction and seal it.

Take 6 µl of the [CuSO4 + BTTAA + sodium ascorbate] master mix and ad it directly into the 24 µl [oligonucleotide + azide] mix.
Pipette up and down so the DMSO dissolved reagents properly mixes with the aqueous reagents. 
Gas the Eppendorf tube and seal it with parafilm.

⏰ Incubate overnight 🌃 🛌 , avoid exposure to direct light ☀️.

🍚 Purification

Select purification method depending on required purity as well as the length of the product you want to isolate.
 If the product is ≥ 16 nt then use the standard Oligo Clean & Concentrator spin column from Zymo.
 If oligo length is ≥ 8 nt but <16 nt then use PAGE purification followed by Amicon filter cleanup.

 🐢 PAGE purification

 Set up the TBE-Urea gel (10% or 15%) to run in 1x TBE buffer. Use a syringe to clean each well.

 Run the gel for 1h at 300 Volt before loading the gel. This helps even the temperature in the gel and help denaturing. 

 Load a maximum of ~1 µg per well (for 9mer ssDNA mw ~2718.6 g/mole).
    The molecular weight, mw, of an oligo can roughly be approximated by its length, nt: mw = 308.95×nt - 62

 Load 2-10 µl per well and run the gel until bands are clearly distinguishable to be cut out.

 Use a razor blade to cut out the upper 1/2 to 2/3 of the band of interest.

⛏️💥 💦 'Crush-and-Soak' recovering 🎣 of oligonucleotides from Polyacrylamide Gel 🍮

 Transfer the gel slices to a microcentrifuge tube. Load a maximum of 1 nmol of 9mer into a single well (check mass with Oligo Calculator).
     Use a sterile glass rod to crush the slice against the wall of the tube.

 Add 500 µl of 0.1 M sodium acetate (pH 6.0).

 Incubate at 90°C for 5 min.

 Push the tube down onto dry ice (-70°C), or into a -80°C freezer. Incubate for 5 min.

 Thaw and transfer to an Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa cut-off).

 Spin for 30 min at 14,000 x g.

🐇 Oligo clean & concentrator spin column

✋ Notes 🛑❗

If it is the first time you use the kit add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA Wash Buffer concentrate (D4060)
or 192 ml 100% ethanol (208 ml of 95% ethanol) to the 48 ml DNA Wash Buffer concentrate (D4061).

The maximum capacity for the column is 5 µg. If you want to run more just run the same column severl times.

 If the reaction is 30 µl add 20 µl of nuclease-free water to the 30 µl reaction mixture for a total of 50 µl.

 Add 100 µl Oligo Binding Buffer to 50 µl sample.

 Add 400 µl ethanol (95-100%) and mix well by pipetting.

 Transfer the 450 µl sample to the Zymo spin column.

 Add 750 µl DNA Wash Buffer to the column and centrifuge for 1 min at 10,000-16,000 x g.

 Transfer the column to a new Eppendorf tube.

 Add ≥ 6 µl elution buffer (water or TE) to the center of the column.

 Centrifuge for 1 min at 10,000-16,000 x g.

The flow-through can immediately be used or stored frozen.
Measure the yield on NanoDrop and then elute further to desired stock concentration or use a SpedVac for lyophilization to make a pellet.