Reaction of AGT with oligodeoxynucleotides

Summary

1. Label oligodeoxynucleotide with [³²P]-ATP.

2. Anneal with complementary strand

3. react with AGT

4. Monitor reaction with ion-exchange HPLC.

5. Determine rate constants

[5¢-³²P]-Labeling of oligodeoxynucleotides.

Follow protocol to label 50 to 300 pmol of O⁶-alkylguanine containing oligo.  

Anneal

Add 50% excess of complementary strand.

Heat at 90 °C for 2 min and then allow to cool slowly to room temperature. 

Reaction with AGT. 

Add AGT to a 1 mL solution of ³²P-oligodeoxynucleotide duplex in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, 5% glycerol at 37 °C.  Typical concentrations will be 5 nM AGT and 1 nM oligodeoxynucleotide. 

Aliquots (100 mL) are removed at various times (0, 10, 20, 30, 60, 120, 240, 360, 600 s) and added to 500 mL 0.1 N NaOH.  The vials are left on Dry Ice.

Store solutions at -78 °C.

Monitor reaction by anion-exchange HPLC.

Column:  Nucleopac Pa-100 (4.6 x 250 mm) from Dionex. 

Buffer A:  10 mM NaOH, 0.4 M NaCl, 0.5 % acetonitrile.

Buffer B:  10 mM NaOH, 1.0 M NaCl, 0.5% acetonitrile.

This gradient works for 15-mer with O⁶mG.  The gradient of choice should be determined at time of reaction.  Inject the basic sample directly onto column.  Retention time of G-oligo was 28 min, O⁶mG-oligo was 21 min. 

Monitor reaction with radioactivity detector.  Can use a ³²P-cell or scintillation cocktail with a tritium cell.  Use a high salt cocktail. 

Care of column:  The column is a polystyrene support with a tertiary amino group.  It is stable at pH above 10 only if salt is present.  Always keep at least 100 mM NaCl in column when base is present.  Wash with 50 mM NH₄OH overnight.  Over weekend store in 50 mM NH₄OH.  Store for longer periods with 50% methanol.

Care of HPLC.   Make sure that the pumps are washed with lots of water.  At least 10 min of 5 mL/min wash at end of day.

Determine` rate constants

Get time course of the conversion of O⁶mG-oligo to G-oligo.  Equation 1 is the reaction in which G and O⁶mG represent oligodeoxynucleotides containing the methylated and demetylated substrate.  The rate equation is shown in equation 2.  Integration of equation 2 produces equation 3 in the [G] is represented by equation 4.  A plot of time as the X-variable and the entire right side as the Y-variable will yield a slope that is equal to k, the second-order rate constant. 

AGT + O6mG → Me-AGT + G	(1)
rate = k2 [AGT] [O6mG-oligo]	(2)
	(3)
	(4)

Buffers

 10 mM NaOH, 0.4 M NaCl, 5% acetonitrile

 Mix together in approximately 900 mL distilled H₂O.

After NaCl dissolves, fill to 1 L.

Filter with 0.22 mm filters

10 mM NaOH, 1 M NaCl, 5% acetonitrile

 Mix together in approximately 900 mL distilled H₂O.

After NaCl dissolves, fill to 1 L.

Filter with 0.22 mm filters

50 mM NH₄Cl

Mix together in 500 mL distilled H₂O.

Filter with 0.22 mm filters