Protocol for the Purification and Synthesis of 15N- and 13C- Labeled Ribonucleotide Triphosphates for Heteronuclear Multidimentional NMR Spectroscopy of RNA

References

Protocol Outline

I. E. Coli Cell Growth
II. Cell Lysis
III. Nucleic Acid Hydrolysis/Separation of rNMPs from dNMPs
IV. Enzymatic Phosphorylation
V. in vitro Transcription using T7 RNA Polymerase

I. E.coli Cell Growth

  1. Solutions & Materials
    1. 1M phosphate buffer solution, pH 7.0 (10x stock)
      • make 1 liter; pH to 7.0 with KOH- start with 850 mL sddH2O
      • KH2PO4 - 136.09 g/L
      • or alternatively, a combination of K(Na)H2PO4/K(Na)2HPO4

    2. Carbon Source = Glucose
      • for 15N-labeling, use 10 g glucose per liter of cell growth
      • for 13C/15N-labeling, make a 20% 13C-glucose solution
        • 1 g of 13C-glucose (99%) per liter of cell growth
        • dissolve 1 g in 5 mL sddH2O

    3. 20% 15N-NH4Cl
      • for 15N-labeling, use 1 g per liter of cell growth
      • for 13C/15N-labeling, use 0.3 g per liter of cell growth

    4. 1M MgSO4 (1000x stock)
      • MgSO4×H2O - 246.37 g/L; make 100 mL

    5. Micronutrients Solution 1 (1000x stock), make 100 mL
      • 1.5 g CaCl2×H2O
      • 3 g Na2EDTA
      • 2.5 g FeCl3×6H2O

    6. Micronutrients Solution 2 (10000x stock); make 100 mL
      • 2.4 g CuSO4×H2O
      • 1.8 g MnSO4×H2O
      • 0.27 g ZnSO4×H2O
      • 0.27 g CoCl2

  2. Inoculation Growth: Start a 1 mL inoculation growth (in sterile culture tube) about 24 hours before the large (1L) growth using the solutions detailed above and 1l of E.Coli cells. All growths should be at 37C and shaken vigorously at all times. Use 4 L flasks with baffles or culture tubes with cap unlocked to ensure proper aeration during growth. The inoculation growth is especially important for 13C labeling, to dilute the effect of storing E. Coli cells in 50% 12C-glycerol.

    another option is to grow an intermediate culture of approx. 25 mL to keep inoculation dilutions > 1/100

  3. Large(1L) Growth: use the following recipe 
    1 mL		inoculation growth
100 mL		10x phosphate buffer stock solution
1 mL		micronutrients solution 1 (1000x stock)
100 l		micronutrients solution 2 (10000x stock)
1 mL		1M MgSO4 (1000x stock)
		glucose (amt. depends on type of labeling)
		ammonium chloride (amt. depends on type of labeling)
    (for 15N labeling, add solids: 10 g glucose and 1 g 15NH4Cl)
    (for 13C/15N labeling, add 1 g 13C-glucose and 1 g 15NH4Cl)
    add sterile(autoclaved) distilled deionized H2O to 1 L (approx. 890 mL)

    equilibrate media in shaker/incubator at 37 C before adding cells grow culture overnight for about 15 hours (solution should be very cloudy) a 1 to 10 dilution will have an O.D. reading of approx. 0.5 @ 600 nm.

  4. Cell Harvest: Harvest cells by centrifugation for 30 minutes at 5000 rpm. Use the GS-3 rotor in the Sorvall (or the JA-10 in the Beckman) centrifuge. Save the supernatant if using 13C-glucose. Pellets may be stored at -20C. Growths should yield approximately 3 (13C/15N labeling) or 6 (15N labeling) grams of wet cells per liter.

II. Cell Lysis

(a general note here: save every fraction from every step in the prep. until the point where an O.D. reading confirms the expected yield)
  1. A. Resuspend the cell pellet in minimal STE buffer (approx. 5 mL per 4 g of wet cells; 0.1 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0)

  2. Add (1/20) volume of 10% sodium dodecyl sulfate. Incubate at 37C for 30 minutes.

  3. Sonicate for 3 minutes using the standard large attachment head (90% output and 50% duty cycle). Repeat 3 times. Cool on ice for 3 minutes after each sonication. Do not use the microtip attacthment.

  4. Preheat (1/2) volume of equilibrated phenol. Add phenol to lysed cells and incubate at 65 C for 45 minutes, during which time the solution is stirred twice for 5 minutes.

  5. Add (24:1) chloroform/isoamyl alcohol in a volume equal to that of phenol added, to give a final ratio of (1:1) aqueous layer to (25:24:1) phenol/chloroform/isoamyl alcohol; vortex. Centrifuge at 5000 rpm (use 150 mL Corex glass tubes , and the GSA rotor in the Sorvall) for 30 minutes to separate aqueous and organic phases. Reserve the aqueous layer (aqueous I). Store the phenol layer if using 13C-glucose.

  6. Back extract the phenol layer with (1/2) volume STE buffer, mixing two layers by stirring for 15-20 minutes. Centrifuge, separate layers, and reserve aqueous layer (aqueous II) as in the previous step. The typical yield for 15N labeling is 4000-5000 OD/L; for 13C, yields are 2000-2500 OD/L (a blender may be used for the back extraction).

  7. If aqueous layers I & II are cloudy, phenol extract (as in Step E.) 1-3 times until a clear solution is obtained (this step will remove any remaining protein & cell components). This step may be done in the clinical centrifuge in 50 mL orange capped tubes.

  8. Take small (500 l) aliquots of aqueous layers I & II, extract once with (24:1) CHCl3/IAA, take a UV scan, and measure the absorbance at 260 nm (make sure the signal is nucleic acid since phenol can have a large absorbance). Calculate the total number of ODs obtained. If the yield looks reasonable, go on to the next step, if it doesnŐt, back extract the phenol layer a second time.

  9. Extract the combined aqueous layers 2-4 times with a half volume of (24:1) chloroform/isoamyl alcohol. Centrifuge at 5000 rpm in the GSA rotor for 15 minutes and keep the aqueous layers. Take a UV scan to verify that phenol has been removed (spectrum should have a maximum at 260 nm). Calculate the total number of ODs obtained. Even if all the phenol hasn't been removed, it will be okay to do the precipitation.

  10. Concentrate the nucleic acids by isopropanol precipitation. Add (1/10) volume of 3M Sodium Acetate, pH 5.4 and 1 volume of isopropanol. Let precipitate at -20 C for 30 minutes. Centrifuge as in the phenol extraction. Pour off the supernatant and take a UV scan to verify that there is no absorbance at 260 nm (the reading at 260 nm may be affected by phenol).

  11. Dry the pellet under vacuum and resuspend in a minimal volume of 1x RNA digest buffer (15 mM Sodium Acetate, 0.1 mM ZnSO4, pH 5.2). Addition of 5mM MgCl2 may help the DNaseI digestion.

III. Nucleic Acid Hydrolysis /Separation of rNMPs from dNMPs

  1. Heat solution in a > 90 C water bath for 10 minutes and then cool in an ice bath.

  2. Add 10 units of P1 nuclease per liter of growth (this amount needs to be optimized according to number of units/OD; start with 1U/25 ODs) and a spatula tip of DNaseI. Incubate at 37 C for 2-3 hours or overnight. Run a mini-gel of the P1 nuclease reaction with an aliquot of undigested nucleic acids before P1 digestion. Use UV shadowing and ethidium staining to monitor the progress of the digestion. If there is any undigested nucleic acid, repeat the denaturing procedure and the P1 nuclease digestion.

    Several diagnostic reactions can be performed with small aliquots including base treatment with KOH @ 55C - RNA is base sensitive whereas DNA is not.

  3. OD the solution and calculate yield. Typical yields are 3000 ODs per liter of growth.

    An anion exchange column step may be inserted here. This would make life easier for the people studying labeled DNA in the lab and would also clean up what gets loaded onto the cis-diol column (and possibly extend its lifetime?).

  4. Cis-Diol Column Preparation
    1. Distill triethylamine (apparatus set up in the Schepartz lab)
    2. Make 1M TEABC, pH 9.5 with 141 mL of distilled TEA and add ddH2O to 1 L. Adjust the pH of the solution to 9.5 with dry ice vapors.

  5. Equilibrate 10 g of Affi-Gel 601 gel (Biorad) in 1M TEABC, pH 9.5 and pour the column. If the column is already poured, pour some TEABC into the column, shake to resettle, and equilibrate the column with approx. 100 mL of 1M TEABC, pH 9.5. Check the pH of the eluent to confirm it is 9.5.

  6. Attach the detector to the chart recorder. Attach cords to the top lead for the blue pen. Set the chart speed to 1 mm/min, mV for the red pen at 10 mV, and mV for the blue pen at 100 mV. Let the TEABC buffer flow until it is just above the column level. Load the NMPs dissolved in TEABC onto the column using a 25 mL pipette. When the sample is just above the column, reattach the tube to TEABC. Add more TEABC so that the buffer level is well above the column bed.

  7. Elute the column with 1M TEABC. The peak coming off the column is the salt and dNMPs. Collect this peak in a separate flask from the other waste and reserve for the people working on labeled DNA in the lab.
  8. Prepare a ddH2O solution between pH 4 and 5 through addition of dry ice. When the first peak has come off the column and the recorder has returned to baseline, elute the rNMPs off the column with ddH2O, pH 4-5. Collect the rNMPs in a round-bottomed flask.

  9. Rotovap the rNMPs and redissolve in ddH2O. Rinse several times to get rid of any TEABC. Measure the absorbance at 260 nm and calculate the yield of labeled rNMPs.

IV. Enzymatic Phosphorylation

  1. Calculate the final volume of the reaction, so that the concentration of rNMPs is 10 mM. Place the undiluted rNMPs in a 50 mL orange cap tube.
  2. Add the following ingredients, per 1 mL of 10 mM rNMPs: 
    100 l			10x reaction buffer (800 mM Tris-HCl, 200 mM
 			KCl, 200 mM MgCl2)
13.3 mmole		Phosphoenol pyruvate (PEP)
0.0067 mmole		Adenosine triphosphate (ATP)
0.011 U			Guanylate Kinase (GK)
2.4 U			Adenylate Kinase (AK)
0.008 U			Nucleotide Monophosphate Kinase (NMPK)
8.0 U			Pyruvate Kinase (PK)
1 mM			DTT
    First add the stir bar, the rNMPs, 10x reaction buffer, and make up the rest of the reaction volume with ddH2O so that the final concentration of rNMPs is 10 mM (for example, if there are 465 mmoles of rNMPs, the total reaction volume is 46 mL). Deaerate the solution for 30 minutes by bubbling N2 through the solution. Adjust the pH of the solution to 6.9 with 1M HCl and 3M KOH. Add all other ingredients.
  3. Monitor the reaction using the HPLC. Run an aliquot of the reaction on the HPLC every 4 hours to see if the reaction has reached completion. Use unlabeled rNMPs and rNTPs as standards. Add more PEP if the reaction has stopped before reaching completion.
  4. After the reaction has reached completion, rotovap the solution to dryness. Resuspend in TEABC and run the Cis-Diol column again. Collect the desired NTPs and rotovap to dryness.
  5. Wash the rNTPs with 50% ethanol and 50% water twice to get rid of any TEABC. Resuspend the rNTPs in DEPC treated water and take an OD at 260 nm.

V. in vitro Transcription with T7 RNA Polymerase

  1. Basic Conditions 
    40 mM 		Tris, pH 8.3 @ rm. temp.
15 mM 		MgCl2 (template-dependent, needs to be optimized)
5 mM 		DTT
1 mM 		Spermidine
.01% 		Triton X-100
16 mM 		labeled rNTPs
200 nM 		DNA template
.075 mg/mL 	T7 RNA Polymerase
50 mg/mL 	PEG 8000
5 U/mL 		Inorganic Pyrophosphatase
  2. Set up the transcription reaction as detailed below. Incubate overnight at 37 C. Add each of the following per 5 mL of transcription: 
    200 l		1M Tris, pH 8.3
75 l		1M MgCl2
25 l		1M DTT
50 l		100 mM Spermidine
50 l		1% (v/v) Triton X-100
		rNTPs (amount dep. on solution concentration)
100 l		Stock DNA Template(11mM T7 promoter/10mM template 
		for RNA of interest)
190 l		2 mg/mL T7 RNA Polymerase
625 l		40% PEG 8000

add sddH2O to make up the volume to 5 mL.
  3. Extract once with 2 volumes of phenol; vortex, spin. Extract 3 times with 0.5 volume of (24:1) chloroform/isoamyl alcohol; vortex, spin. Place the aqueous layer in a new tube for each extraction.

  4. Concentrate RNA by ethanol precipitation. Add (1/10) volume of 2M NaCl precipitate in the clinical centrifuge for 30 minutes. Remove the ethanol supernatant and wash the pellet with 70% ethanol. Dry the pellet using an aspirator.

  5. Desalt the RNA using NAP columns. Use one NAP-25 column for no more than 5 mL of transcription. Equilibrate the NAP-25 column with 25 mL of ddH2O. Load the sample in 2.5 mL. After the sample has been loaded onto the column, elute the RNA with ddH2O, and collect the next 4 mL of eluent in 1 mL fractions.

  6. Speed Vac samples overnight. Resuspend samples in a minimal amount of Urea Loading Buffer. Add a small amount of loading buffer with dyes just prior to loading.

  7. Set up 20% denaturing polyacrylamide gel (1.5 mm, 1x TBE running buffer, 400l 10% APS, 55 l TEMED). Pre-run gel @ 50 W for 30 minutes. Load sample in a minimal volume. Run gel for approx. 6 hours. Visualize the RNA by UV shadowing and cut out the band (a transcript of 27 nucleotides should run just above the xylene cyanol dye).

  8. Electroelute RNA from gel slices using the Elutrap at 225 V. Use 0.5x TBE buffer and enough chambers to accomodate all of the gel slices. Collect fractions every 2 hours and take check the absorbance at 260 nm.

  9. Speed vac the elutrap fractions to reduce the volume for dialysis.

  10. Dialyze RNA against 10x NMR Buffer for 24 hours. Use a dialysis membrane with a molecular weight cut-off of 2,000.

  11. Dialyze RNA against 1x NMR Buffer for 24 hours. Take an absorbance measurement at 260 nm and calculate yield.

  12. Speed Vac sample to an appropriate volume and dialyze against 1x NMR Buffer for 24 hours.

  13. Put sample in an NMR tube and start doing NMR experiments.
marino@carb.nist.gov Last updated 20 March 2000