KMnO₄ Footprinting   

Materials and Reagents

1. Radiolabeled DNA fragment of interest
   
2. 14.3 M β-mercaptoethanol
   
3. 500 mM EDTA
   
4. Phenol
   
5. Bromophenol blue (Sigma-Aldrich, catalog number: BO126 )
   
6. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
   
7. Formamide deionized (Panreac Applichem, catalog number: A2156 )
   
8. Cholorophorm
   
9. Piperidine, purity grade: pro analysis (p.a.) (e.g. Sigma-Aldrich, catalog number: 411027 )
   
10. Ethanol (purity grade: pro analysis) (p.a.)
    
11. X-ray films
    
12. Glycogen (1 μg/μl) (e.g. Roche, catalog number: 10901393001 )
    
13. NaOAc (300 mM pH = 5.5)
    Optional for Procedure B (primer extension analysis)
    
14. Non-radio labeled DNA fragment of interest
    
15. 5'-³²P-labeled desoxyoligonucleotide primer
    
16. NaOH (10 mM)
    
17. Tris
    
18. MgSO₄
    
19. DTT
    
20. Klenow fragment of DNA polymerase I (e.g. Biolabs, catalog number: MO210S )
    
21. NH₄OAc
    
22. 370 mM KMnO₄ stock solution (a 1:1 dilution with H₂O is used for the reaction) (see Recipes)
    
23. Phenol/Chloroform (see Recipes)
    
24. Neutralization solution (see Recipes)
    
25. dNTP mix (see Recipes)
    
26. Stop mix (see Recipes)
    
27. Electrophoresis loading buffer (see Recipes)
    

Equipment

1. Table-top centrifuge
   
2. Vortex shaker
   
3. Incubator
   
4. Polyacrylamide gel electrophoresis system
   
5. Vacuum concentrator
   

Procedure

1. Footprinting with 3'- or 5'-end-labeled DNA analyzed by piperidine-catalyzed strand scission.
   1. Add 4 μl of 160 mM KMnO₄ solution to radiolabeled DNA fragments of interest (40 ng; 5,000 to 10,000 cpm) (in the presence or absence of a binding partner) in a total volume of 40 μl.
      
   2. Incubate samples for 2 min at 30 °C; mix gently.
      
   3. Stop reaction by the addition of 4.8 μl β-mercaptoethanol; mix again.
      
   4. Put samples on ice.
      
   5. Add 5.3 μl 500 mM EDTA.
      
   6. Extract samples three times with 100 μl phenol/chloroform by vigorous shaking on a vortex for 1 min each time.
      
   7. Centrifuge samples (5 min, 3,000 rpm) and take aqueous layer.
      
   8. Precipitate samples with 2.5 volumes absolute ethanol (-20 °C).
      
   9. Centrifuge tubes (10 min, 10,000 rpm, 4 °C).
      
   10. Dissolve wet ethanol pellets in 70 μl of 10% (v/v) piperidine.
       
   11. Incubate samples at 90 °C for 30 min.
       
   12. Lyophilize samples in a vacuum concentrator until dry.
       
   13. Add 30 μl distilled water and lyophilize again.
       
   14. Repeat step 9.
       
   15. Dissolve final pellets in 50 μl distilled water.
       
   16. Add 4 μl 300 mM NaOAc (pH 5.5).
       
   17. Add 1 μl glycogen (1 μg/μl) to aid precipitation.
       
   18. Precipitate samples with 2.5 volumes absolute ethanol (-20 °C).
       
   19. Wash pellets with 150 μl 70% ethanol.
       
   20. Dry samples briefly in a vacuum concentrator to get rid of excess ethanol and dissolve in 5 μl electrophoresis loading buffer.
       
   21. Separate on a 10% or 15 % denaturing polyacrylamide gel, respectively, depending on the size of the DNA fragment.
       
   22. Visualize radioactive bands by autoradiography (over-night X-ray exposure).
       
   
   
2. Footprinting with non-labeled DNA analyzed by primer extension
   1. Add 4 μl of 160 mM KMnO₄ solution to 40 μl non-labeled DNA fragments (~100 ng) in presence or absence of a binding partner.
      
   2. Follow steps 2 to 8 of procedure A.
      
   3. Dissolve pellet in 35 μl distilled water.
      
   4. Add 1 μl of an appropriate 15 to 20^mer 5'-³²P-labeled desoxyoligonucleotide primer complementary to the downstream sequence of interest (~ 5 x 10⁵ cpm). For convenient resolution the primer should not bind farther than 50 to 80 nucleotides downstream to the region of interest.
      
   5. Add 4 μl 10 mM NaOH.
      
   6. Incubate for 2 min at 80 °C.
      
   7. Put samples on ice for 5 min.
      
   8. Add 4.5 μl of neutralization solution and mix samples.
      
   9. Incubate for 3 min at 68 °C for hybridization.
      
   10. Put samples on ice and add 5 μl of all four dNTPs (5 mM each), 1 unit Klenow fragment of DNA polymerase I and mix gently.
       
   11. Extension reaction is carried out for 10 min at 50 °C.
       
   12. Put samples on ice and terminate the reaction by the addition of 17 μl stop mix.
       
   13. Samples are precipitated with 2.5 volumes ethanol (-20 °C).
       
   14. Wash pellets with 150 μl 70% ethanol.
       
   15. Dry samples briefly in a vacuum concentrator to get rid of excess ethanol and dissolve in 5 μl electrophoresis loading buffer.
       
   16. Separate on a 10% or 15% denaturing polyacrylamide gel, respectively, depending on the size of the cDNA fragments expected.
       
   17. Visualize radioactive bands by autoradiography (over-night X-ray exposure).
       

Recipes

1. KMnO₄ stock solution (370 mM)
   KMnO₄ (MW = 158.04) has a solubility limit of ~60 g/L (corresponding to 0.37 M).
   A 200 ml stock solution is prepared by adding 12 g KMnO₄ to 220 ml distilled water. The solution is boiled until it reaches a final volume of 200 ml. This stock solution can be kept in a dark bottle for several months.
   
2. Phenol/Chloroform
   A 1:1 mixture (v/v) of phenol and chloroform, purity grade: pro analysis (p.a.) is used for extraction. The phenol has been saturated with 1 M Tris-HCl (pH 7.9) before and 0.1% (final concentration) 8-Hydroxychinolin should be added to the phenol to avoid oxidation.
   
3. Neutralization solution
   0.5 M Tris-HCl (pH 7.2)
   0.1 M MgSO₄
   2 mM DTT (optional for procedure B)
   
4. dNTP mix
   5 mM each, dATP, dCTP, dGTP, dTTP (optional for procedure B)
   
5. Stop mix
   4 M NH₄OAc
   20 mM EDTA (optional for procedure B)
   
6. Electrophoresis loading buffer
   0.1% (w/v) bromophenol blue
   0.1% xylene cyanol
   95% formamide deionized
   25 mM EDTA (pH 8.0)
   

Acknowledgments

We like to thank people from the laboratory for helpful discussions. Work from this laboratory was funded by the Deutsche Forschungsgemeinschaft (DFG) SPP 1258 [Wa455/13-2] and [PU 435/1-1].

References

1. Hayatsu, H. and Ukita, T. (1967). The selective degradation of pyrimidines in nucleic acids by permanganate oxidation. Biochem Biophys Res Commun 29(4): 556-561.
   
2. Stratmann, T., Pul, U., Wurm, R., Wagner, R. and Schnetz, K. (2012). RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants. Mol Microbiol 83(6): 1109-1123.
   
3. Sasse-Dwight, S. and Gralla, J. D. (1989). KMnO₄ as a probe for lac promoter DNA melting and mechanism in vivo. J Biol Chem 264(14): 8074-8081.