Materials

Caution: small-molecule DNA-damaging agents are known or suspected carcinogens and display high-toxicity profiles. Universal precautions (e.g., proper containment and disposal, gloves, eye protection, lab coat, and others) should be insured at all times.

1. DNase I buffer: 40 mM Tris-HCl, 10 mM MgSO4, 1 mM CaCl2, pH 8.0.

2. BamHI buffer: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), pH 7.9.

3. Buffer for Enediynes: 40 mM Tris-HCl, pH 7.5.

4. Buffer for Bleomycin: 40 mM potassium phosphate, pH 7.5.

5. Buffer for iron (II) chelators: 40 mM Tris-HCl, pH 7.5, with sodium ascorbate and hydrogen peroxide (see Subheadings 3.6. and 3.7.).

6. All oligonucleotides were dissolved and stored in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). To prevent particulate matter, all buffers must be ultra-filtered (<0.45 |m noncellulose membrane) before use.

7. FluoroMax-2 spectrofluorometer equipped with DataMax™ for Windows (Instruments SA; Edison, NJ) and the temperature controlled by a Haake DC10 circulator.

8. Suprasil quartz cuvet (10-mm path) fitted with a magnetic stirring bar in a total volume of 2 mL.

9. FLUOstar OPTIMA (BMG Labtech; Durham, NC) 96-well plate reader (Software v1.20-0) equipped with filters (absorbance = 485 nm, emission = 520 nm) conducive for the standard green fluorescence assay. Reaction conditions, using opaque polystyrene Costar 96-well plates, are identical to the FluoroMax protocol, albeit at one-tenth the volume (200 | L total).

10. MBL design: we constructed two different break-light probes (purchased from GIBCO/BRL) for the designed experiments: "break light A" (BLA) contains the known calicheamicin recognition sequence (5'-TCCT-3'); "break light B" (BLB) contains the BamHI recognition sequence (5'-GGATCC-3') with a required 3-bp overhang (see Fig. 2C). The loop of both probes consisted of a T4 loop to ensure nonhybridizing interactions. The 5'-fluorophore of both probes was fluorescein (absorbancemax = 485 nm, emissionmax = 517 nm), whereas the corresponding 3'-quencher was 4-(4'-dimethylaminophenylazo) benzoic acid (dabcyl). Assays were prepared from stock solutions (100- and 1000-fold break light dilutions, in TE buffer). An oligonucleotide identical to BLB lacking both fluorescein or dabcyl ligations was purchased for titrations within the BamHI kinetic studies (see Subheading 3.2.).

11. DNase I and BamHI were purchased from Promega (Madison, WI).

12. EDTA and methidiumpropyl-EDTA were purchased from Sigma (St. Louis, MO).

13. Enediynes employed in this study are not commercially available and were gifts from Wyeth (calicheamicin Yj'; Pearl River, NY) and Bristol-Myers-Sqibb (esperamicin A1; Wallingford, CT). Enediyne stocks were prepared in 50:50 methanol/glycerol at 10 mg mLJ, as determined by optical standardization (e>26J = 75,000 MJcmJ; ref. 8).

14. Blenoxane® (a mixture containing approx 70% bleomycin A2 and 30% bleomycin B2; Mead-Johnson, Princeton, NJ) was dissolved in deionized water and optically standardized (e>29J = 17,000 MJcmJ; ref. 9).

15. All iron (Il)-containing solutions were prepared fresh from (NH4)2Fe(SO4)2 daily with 1 mM H2SO4 to prevent hydrolysis and oxidation (10).

3. Methods

3.1. General Considerations

1. Cleavage reactions are initiated at time (t) = 0 by the addition of enzyme or activator (e.g., DTT for 1).

2. Maximum fluorescence (Fmax) for a given break light concentration in each assay system is accomplished by a replica assay containing saturating DNase. Fmax is correlated to the amount of break light being used ([BL]) by the Eq. 1:

Example: Maximum fluorescence of a break light concentration of [BL] = 10 nM was Fmax = 10,000 relative fluorescence units (RFU). Therefore, the conversion is (5 nM/10,000 RFU) or 0.001 nM (or 1 pM) per RFU. That is, each RFU represents 1 pM of break light cleavage (see Note 1).

3. Pseudo first-order kinetics are determined through Eq. 2 where [A]t is the concentration of break light at time (t) and [A]0 is the initial break light concentration used within the reaction. Least squares analysis gives the slope (k), or rate, which was converted to the velocity (V) by Eq. 3. The maximum velocity (Vmax) is then selected from the range of concentrations examined (see Note 2).

3.2. Proof of Principle: Enzymatic Cleavage of Break Lights

1. Incubate 3.2 nM BLA or BLB in DNase I or BamHI buffer and equilibrate.

2. Initiate reaction by the addition of enzyme (100 U BamHI or 10 U DNase I).

3. Incubation proceeds over 10-min time base scan.

4. Results: DNase I-induced cleavage provides nonspecific cleavage to both BLA and BLB, where as BamHI induces cleavage only upon BLB, whose DNA recognition site within the break light stem is specific toward the BamHI endonuclease (see Fig. 3).

3.3. BamHI Steady-State Kinetics

1. Incubate 3.2 nM BLB in BamHI buffer and equilibrate.

2. Initiate reaction by the addition of 10 U BamHI.

3. Incubation proceeds over 15-min time base scan.

4. Steps 1-3 are repeated each with the added concentration of BLB oligonucleotide lacking the fluorophore and quenching moieties (0, 3.8, 7.7, 38.5, 77.0, 192.5, and 385 nM).

5. As the carrier dilution by nonlabeled oligonucleotide alters the apparent rate of DNA scission, the actual velocity (Vact) is calculated from Eq. 4:

where Vobs is the observed velocity and Sact and S* are the total substrate concentration (break light plus the unlabeled oligonucleotide) and break light concentration, respectively (11).

6. Data from Eqs. 2-4 are used toward elucidation the Michaelis-Menten parameter Km and turnover rate (Vmax/[agent], also known as kcat).

Fig. 3. The demonstration of molecular break light specificity and general proof of principle. The observed change in fluorescence intensity over time of an assay containing 3.2 nM break light at 37°C. (A) "Break light A" (BLA) with 100 U of BamHI (open squares), "break light B" (BLB) with 100 U of BamHI (open circles), and BLB without enzyme (blocked circles). (B) BLA with and 10 U of DNase I (open sqaures), BLB with 10 U of DNase I (open circles), and break light A without enzyme (blocked circles). (Reproduced with permission from ref. 2.)

Fig. 3. The demonstration of molecular break light specificity and general proof of principle. The observed change in fluorescence intensity over time of an assay containing 3.2 nM break light at 37°C. (A) "Break light A" (BLA) with 100 U of BamHI (open squares), "break light B" (BLB) with 100 U of BamHI (open circles), and BLB without enzyme (blocked circles). (B) BLA with and 10 U of DNase I (open sqaures), BLB with 10 U of DNase I (open circles), and break light A without enzyme (blocked circles). (Reproduced with permission from ref. 2.)

3.4. Enediyne-Dependent Scission

1. Incubate 3.2 nM BLA in 40 mM Tris-HCl, pH 7.5 with varying enediyne concentration (0.15, 0.31, 0.78, 1.6, 3.2, 15.9, and 31.7 nM) and equilibrate.

2. Initiate reaction with 1 pL 100 mM DDT (50 ||M total concentration).

3. Incubation proceeds over 10-min time base scan.

4. Pseudo first-order kinetics are determined using Eqs. 2 and 3.

5. Note: two distinct rates are observed from enediyne reductive activation (0-50 s) and maximal enediyne-based cleavage (Vmax; 50-200 s).

3.5. Bleomycin-Dependent Scission

1. Incubate 3.2 nM BLA in 40 mM potassium phosphate buffer, pH 7.5 with varying bleomycin concentrations (9.5, 19, 47.5, 95, 142.5, and 190 nM) and equilibrate.

2. Inititate reaction with the addition of 65 mM iron (II).

3. Incubation proceeds over a 5-min time base scan.

4. Pseudo-first order kinetics are determined using Eqs. 2 and 3.

3.6. EDTA-Fe(II)-Dependent Cleavage

1. Incubate 33.8 nM BLB in 40 mM Tris-HCl, pH 7.5, 2.5 mM sodium ascorbate, 0.0075% hydrogen peroxide, and equilibrate.

2. Inititate cleavage with the addition of EDTA-Fe(II) in a 2:1 ratio. Final iron concentrations: 1.3, 3.1, 6.3, 12.5, 31.3, and 125 |M.

3. Incubation proceeds over 10-min time base scan.

4. Pseudo first-order kinetics are determined using Eqs. 2 and 3.

3.7. Methidiumpropyl-EDTA-lron (ll)-Dependent Cleavage

1. Incubate 3.2 nM BLB in 40 mM Tris, pH 7.5, 2.5 mM sodium ascorbate, 0.75 ppm hydrogen peroxide, and equilibrate.

2. Initiate cleavage with the addition of EDTA-Fe(II) in a 1.2:1 ratio. Final iron concentrations: 0.13, 0.25, 0.50, 1.5, 2.5, 5.0, 10 |M.

3. Incubation proceeds over 10-min time base scan.

4. Pseudo first-order kinetics are determined using Eqs. 2 and 3.

4. Notes

1. The previously mentioned methods are performed under the assumption of 100% cleaving efficacy of the scission agents. Because of the high-efficiency of the fluorophore-quencher pair, it is not possible to quantitate the amount of uncleaved break light. Therefore, this procedure should be used in conjunction with an high-performance liquid chromatography or polyacrylamide gel electrophoresis analysis of the reaction mixture to quantitate the true amount of cleaved oligonucleotide (12). The data can then be adjusted accordingly to ascertain the kinetic parameters as previously outlined.

2. We compared the turnovers of the nonenzymatic cleavage by small molecules with the turnover/£cat of enzymatic cleavage with the caveat that the small molecule-based cleavage is a single turnover event (8).

References

1 . Klostermeier, D. and Millar, D. P. (2002) Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids. Biopolymers 61, 159-179.

2 Brodie, S., Giron, J., and Latt, S. A. (1975) Estimation of accessibility of DNA in chromatin from fluorescence measurements of electronic excitation energy transfer. Nature 253, 470-471.

3 Yarbrough, L. R., Schlageck, J. G., and Baughman, M. (1979) Synthesis and properties of fluorescent nucleotide substrates for DNA-dependant RNA polymerases. J. Biol. Chem. 254, 12,069-12,073.

4 Wu, F. Y-H. and Tyagi, S. C. (1987) Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. J. Biol. Chem. 262, 13,14713,154.

5 Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckett, D. R., Diekmann, S., and Lilley, D. M. J. (1989) Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature 341, 763-766.

6 Tyagi, S. and Kramer F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303-308.

7 Biggins, J. B., Prudent, J. R., Marshall, D. J., Ruppen, M., and Thorson, J. S. (2000) A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases. Proc. Nat. Acad. Sci. USA 97, 13,537-13,542.

8 Myers, A. G., Cohen, S. B., and Kwon, B.M. (1994) A study of the reaction of calicheamicin yj with glutathione in the presence of double-stranded DNA. J. Am. Chem. Soc. 116, 1255-1271.

9 Burger, R. M., Horwitz, S. B., and Peisach, J. (1985) Stimulation of iron(II) bleomycin activity by phosphate-containing compounds. Biochemistry 24, 36233629.

10 Burger, R. M., Projan, S. J., Horwitz, S. B., and Peisach, J. (1985) The DNA cleavage mechanism of iron-bleomycin. Kinetic resolution of strand scission from base propenal release. J. Biol. Chem. 261, 15,955-15,959.

11 Roy, K. B., Vrushank, D., and Jayaram, B. (1994) Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants KM and kcat for BamHI restriction endonuclease: an empirical and iterative approach. Anal. Biochem. 220, 160-164.

12 Hashimoto, S., Wang, B., and Hecht, S. M. (2001) Kinetics of DNA cleavage by Fe(II)-bleomycins. J. Am. Chem. Soc. 123, 7437-7438.

13 Jacobsen, E. N. and Finney, N. S. (1994) Synthetic and biological catalysts in chemical synthesis: how to assess practical utility. Chem. Biol. 1, 85-90.

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