Bloconlugate Chem. 1003, 4, 69-77
60
Nuclease Activity of 1,lO-Phenanthroline-Copper. New Conjugates with Low Molecular Weight Targeting Ligands Chi-hong B. Chen, Abhijit Mazumder, Jean-Francois Constant, and David S. Sigman' Department of Biological Chemistry, School of Medicine, Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90024. Received September 2, 1992
The chemical nuclease activity of 1,lO-phenanthroline-copperdepends on DNA sequence because the coordination complex has affinity for DNA. In order to target this efficient nucleolytic activity, it is essential to override its inherent specificity. The minimal size of ligands capable of redirecting the specificity has been investigated. A conjugate (HOP) prepared by alkylating Hoechst dye 33258 with 5-(iodoacetamido)-l,l0-phenanthrolinehas a greater preference for A-T-rich regions than the unsubstituted 1,lO-phenanthroline-copper complex, reflecting the specificity of this A-T-specific minorgroove binder. However, since quatemizingthe dye with 5-(iodoacetamido)-l,lO-phenanthrolineincreases its affinity for DNA, the specificity of cleavage by the conjugate is less than the binding selectivity of the dye. Linking 1,lO-phenanthroline with the peptide of the helix-turn-helix domain of the Trp repressor specificity results in a conjugate with greater reactivity for the operator sequence than the unsubstituted complex. The intrinsic affinity of the 1,lO-phenanthrolindu can only be partially overridden by the conformationally unstable peptide. Attachment of 1,lO-phenanthroline to a deoxyoligonucleotide complementary to a single-stranded loop of RNA successfully targets the scission of the chemical nuclease. Cleavage sites are observed not only contiguous to the site of hybridization but also at nonadjacent sequence positions. The latter set of sites must be close in space to the 5' end of the hybridized deoxyoligonucleotide.
INTRODUCTION
1,lO-Phenanthroline-copperion in the presence of thiol and hydrogen peroxide efficiently nicks DNA and RNA by oxidative attack on the (deoxy)ribose moiety (1,2). The kinetic mechanism involves the following sequence of steps (eq 1): (a) the reversible binding of the tetrahedral (OP)~CU++ DNA
Kd
(OP)~CU+-DNA
(1)
/? scission products
2:11,1O-phenanthroline-cuprouscomplex to DNA or RNA (3); (b) the one-electron oxidation of the nucleic acidbound coordination complex (4, 5), (c) attack by the oxidative species generated on the (deoxy)ribose (6, 7), and (d) a series of elimination reactions leading to strand scission (8). Recent studies have demonstrated that ascorbic acid activates the scission chemistry more efficiently than mercaptopropionic acid, which may decrease the concentration of the reactive 2:l 1,lO-phenanthroline-cuprous complex (9, IO). The second phenanthroline converts the 1:l complex to an efficient nucleolytic agent because it generates a hydrophobic cation with high affinity for DNA and RNA ( 4 ) . Substitution at the 5-position of phenanthroline of functionalities such as phenyl, methyl, bromo, nitro, amino, acetamido, and glycinamido have modest effects on the rate of scission of DNA. The scission patterns obtained for copper complexes of these phenanthroline derivatives are comparable to those obtained with the copper complex of the unsubstituted phenanthroline. Variations in the
* Address correspondence to this author at the Molecular Biology Institute.
rate of scission can be attributed to the stability of the noncovalent complex or differences in the rate of oxidation of the cuprous complex by hydrogen peroxide (3). In contrast, the copper complexes of 1,lO-phenanthrolines derivatized a t the 2 position with a methyl, carboxyl, carboxamide, or carbinol group do not cleave DNA. The lack of reactivity is due to a slow rate of oxidation of the cuprous complex attributable to steric hindrance involving the ortho substituents in the square planar cupric complexes (3). In fact, 2,9-dimethyl-l,lO-phenanthroline quenches the scissionreaction because it sequesters copper ion as the redox-stable cuprous complex (5). The specificity and the nuclease activity of 1,lOphenanthroline-copper has been altered by conjugating the ligand at its 5-positionto proteins, peptides, andnucleic acids (2). The most general method for the preparation of the bioconjugates has been the alkylation of sulfhydryl groups of the carrier with 5-(iodoacetamido)-l,lO-phenanthroline (11-15). Coupling 5-glycinamido-1,lO-phenanthroline to the terminal phosphates of an oligonucleotide has also been successful (16). If the carrier ligand has high affinity for one aspect of nucleic acid structure, the reactivity of the bioconjugate will reflect its binding specificity and not that of the hydrophobic cation, 1,lOphenanthroline-copper. The minimal size, conformational stability, and specificity of carrier ligands which are capable of redirecting the intrinsic scission specificity of the nuclease activity of 1,lO-phenanthroline-copper(OP-Cu) have not been defined. They have been investigated in the present report because OP-Cu has significant advantages relative to ferrous EDTA as a scissionreagent. Unlike ferrous EDTA, which has been widely used to accomplish the chemical cleavage of the phosphodiester backbone, OP-Cu does not generate diffusible hydroxyl radicals. Therefore nonlabeled DNA or tRNA does not have to be included in reaction mixtures as a free-radical trap to visualize site-
1043-18Q2/93/29Q4-QQ698Q4.QQ/Q 0 1993 American Chemical Society
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Bioconjugate Chem., Vol. 4, No. 1, 1993
Chen et al.
specific scission. Avoiding these extraneous nucleic acids would be essential for a range of applications for chimeric cutters. However, the problem with linking OP-Cu to any carrier is that it is not clear upon conjugation of 1,10-phenanthroline to a ligand whether the specificity of 1,lO-phenanthroline or its carrier will dominate or whether the conjugate will demonstrate an entirely new specificity arising from the influence of the binding elements on one another. The goal of this study was to address this issue by examining the reactivity of bioconjugates prepared by tethering 1,lO-phenanthrolineto carrier ligands of modest affinity and specificity for different aspects of nucleic acid structure. These included (a) Hoechst dye 33258, a fluorescent cytologicalstain with specificity for the minor groove of A-T-rich regions of DNA (17-19); (b) the helixturn-helix of a peptide derived from the Escherichia coli Trp repressor, a protein which had previously been converted into a site-specific DNA scission reagent (11); and (c) a heptamer 5’-ACTGCCT-3’- which is complementary to a single-stranded loop of a lac-operon-derived RNA fragment (20). The structures of conjugatesprepared from Hoechst 33258 (HOP), the helix-turn-helix peptide (P-2),and the heptamer (OP-5’-ACTGCCT-3’) are indicated below. PH
HOP H
0
I
R;-C-‘~~-N
-P I I -0-ACTGCCT
c,
II
0 N
d 0 P-heptamer
q g
0
H
II
I
NTFHp’S’CH2’CH .‘.QRELKNELGVGlATITRWSN-amide N-H I
N,
o= c’
\
(14C)CH3
P-2
The synthesis of HOP was prompted by an interest to target (or restrict) the scission chemistry of 1,lO-phenanthroline-copper to A-T-rich regions. Although A-T-rich regions are effectively cleaved by the unsubstituted reagent, it also attacks G-T-rich sequences as well (21, 22). In view of the use of Hoechst 33258 as a fluorescent stain for metaphase chromosomes (23),a scission reagent that reflected the binding specificity of the dye to specific chromosomal regions could prove useful in mapping experiments. P-2 was studied to determine if the helix-turn-helix peptide of theE. coli Trp repressor linked to OP-Cu would exhibit similar scission to the intact protein which had been converted into a site-specificnuclease by modification of lysyl residues with 1,lO-phenanthroline (11).
The reactivity of 1,lO-phenanthroline-copperlinked to short deoxyoligonucleotides (up to seven bases long) complementary to single-stranded loops of RNA has not been previously studied. An earlier study (24) had demonstrated that a deoxyoligonucleotide linked to phenanthroline at the 5’ end and complementary to sequence positions 1-21 of a lac mRNA fragment cleaved i b target at sequence positions 20-25 after denaturation and annealing. Potential applications of short 1,lOphenanthroline-linked conjugates would be to use the scission pattern to identify nucleotides adjacent in space to a single-stranded loop of native RNAs. EXPERIMENTAL SECTION
HOP. Synthesis. A 100-mg portion of Hoechst 33258 (0.236 mmol) was suspended in 1mL of dry DMF under a nitrogen atmosphere. The resulting solution was heated at 75 “C and then triethylamine (100 pL, 0.717 mmol) was added to obtain the free base form of the dye. Then, 5-(iodoacetamido)-l,l0-phenanthroline (25)(100mg, 1.24 times excess) was added and the reaction allowed to proceed for at least 6 h at 75 “C. The mixture is then analyzed on TLC (silica gel) by using successively two different solvents. In the first solvent, pure methanol, Hoechst 33258, and 5-(iodoacetamido)-OP moved close to the solvent front, but the product HOP moved only slightly. In the second solvent, MeOH/NH40H 95/45, HOP moves significantly (Rf about 0.15). More NH4OH increases the Rf but causes smearing. The product can be purified by paper chromatography using 3M paper and 2-propanol, H20, and NH4OH (8/1/1). The slowest fluorescent band (Rf = 0.13) was cut out, and the paper diced into small pieces and covered with 20% acetic acid with heating at 65 “C for 1h. The products are isolated after filtering on Celite and evaporating to dryness. Alternatively, the product can be purified by silica gel chromatography (methanoVNH40H 8/21. It exhibited fluorescence characteristic of the dye and formed a deep red color with ferrous ion typical of the phenanthroline nucleus. The structure of the product was supported by fast-atom bombardment mass spectrometry [FAB MS: mle 660 (M+)], NMR [1H NMR (360 MHz, TFA-d): 6 9.45-8.00 (m, 12 H, aromatics), 7.35 (d, J = 9 Hz, 1 H, 33258),5.25 (e, 2 H, CH2N+),4.90 (t,2 H, 2 NH), 4.40-4.80 (m, 8 H, 4 CH2), 4.10 (s, 4 H, PhOH), 4.00 (8, 3 H, CH3)1, and UV-vis spectrometry. Dye concentration can also be determined spectroscopically in 5 mM HEPES, 100 mM sodium chloride, pH 7.0. The extinction coefficient is 4.2 X lo4 M-l cm-’ at 338 nm. Isolation and Labeling of Lac UV-5and pm Terminator Fragment. Conditions for the preparation of the lac UV-5 and pm terminator fragment have been previously described (26,27). Scission Conditions for HOP-Cu and OP4u. A solution containing 2.5 pM HOP and 0.5 pM Cu2+ was diluted to concentrations of HOP ranging from 0.05 to 0.01 pM. The scission chemistry was activated by the addition of 2.9 mM 3-mercaptopropionic acid (MPA), carried out for 20 h at 37 “C, and then quenched by the addition of 2,9-dimethyl-l,lO-phenanthroline. The data for unsubstituted 1,lo-phenanthroline-Cu2+ was obtained using 5 pM OP and 1.2 pM Cu2+. This reaction was initiated by addition of 2.9 mM MPA, incubated for 1.5 h, and then quenched with 2 mM 2,9-dimethyl-l,lOphenanthroline. After ethanol precipitation, each sample was redissolved in loading buffer composed of 80% deionized formamide, 0.1 3’% bromophenol blue, 0.1 % xylene cyanol, and 1mM EDTA and analyzed on a 10%
Nuclease Actlvky of 1,1O-Phenanthrollne-Copper
Bi0con)wte Chem., Vol. 4, No. 1, I993 71
polyacrylamide/8.3 M urea sequencing gel. Exposure of the resulting gel to X-ray film produced the autoradiogram displayed. To obtain footprints of Hoechst 33258,5 pmol of endlabeled lac operator DNA in 20 pL of 50 mM Tris-Ac (pH 8.0), 1.0 mM MgC12, and 100 mM KC1 were incubated with 10 pM of the DNA ligand for 30 min at 22 "C in the dark. The footprinting reaction was initiated by adding 200 pM OP and 45 pM CuSO4 following by 5.8 mM 3-mercaptopropionic acid. Peptide Derived from the Helix-Turn-Helix of the Trp Repressor. The peptide synthesized (P-2)has the following sequence: 77 OP-K['4C]acetyl-Cys-GIn-Arg-Glu-Leu-Lys-Asn-Glu-Leu-Gly-~-Gly-Jle05 Ala-Thr-lle-Thr-Arg-Trp-Ser-Asn-CONH2
-
It was synthesized on an Applied Biosystem Peptide Synthesizer using standard solid-phase methodology (28). The N-terminuswas acetylated with ['%]acetic anhydride. P-2 (5 mg) was dissolved in 2 mL of phosphate buffer (0.05 M, pH 8, containing 10 mM 0-mercaptoethanol). After standing overnight at 4 "C, the peptide was separated from 0-mercaptoethanol by passage through a G-25 spin column. Tritiated 5-(iodoacetamido)-l,l0-phenanthroline (250 pL of 4 mg/mL, 0.1 mCi/mmol) was added to the eluant of the spin column. The reaction mixture was incubated for 3-4 h at 4 "C, concentrated on the speed vac, and then redissolved in water (final volume of 1mL). Peptide was separated from excess 5-(iodoacetamido)-l,10-phenanthroline by two passes through a spin column. The modification of the peptide was assayed by HPLC analysis on a reverse-phase (2-18 column and the l4W3H ratio of the collected eluant. Footprinting and Cleavage Conditions for 1,lOPhenanthroline-Derivatized Trp Repressor and Derived Peptides. A solution of 3'4abeled aroH fragment (11) (0.01-0.05 pmol), 1,lO-phenanthroline-derivatized peptide (5,25, and 100 pM), and L-tryptophan (10 mM) was incubated at room temperature for 5 min in 10 pL of Tris-HCl(30 mM, pH 8-01,KC1 (100 mM), and MgClz (3 mM). The cleavage reactions were initiated by adding 1 pL of CuS04(200 pM) and 1pL of MPA (58mM) and then incubated for 2 h at 37 "C. The reactions were quenched by 2,9-dimethyl-l,lO-phenanthroline.In each case, 1,10-phenanthroline-derivatizedTrp repressor (100nM) was used as a positive control for the peptide-targeted scission reaction. Targeted Scission of RNA. RNA substrates derived from the E. coli lac operon were prepared and 3'4abeled as described previously (20). Purification of an Heptameric Deoxyribooligonucleotide. The heptamer of sequence 5'-ACTGCCT-3' was synthesized on an automated DNA synthesizer. The 5'terminal trityl group was not removed so that the oligonucleotide could be purified on a C18 reversed-phase HPLC column. After equilibration of the column with 85% 50 mM triethylammonium acetate, pH 7, and 15% acetonitrile, the deoxyoligonucleotide was eluted with a linear gradient from 155% to 35 % acetonitrile over 12 min. The solution containing the heptamer was evaporated to dryness and redissolved in 1 mL of 80% acetic acid to remove the dimethoxytrityl group. Synthesis of 5'- (5-Glycinamido-1,lO-phenanthroline)-ACTGCCT. This product was prepared using the water-soluble-carbodiimidemethod of Chu et al. (29).This procedure had been previously used in the synthesis of a
1,lO-phenanthroline derivative of a 21 base long deoxyoligonucleotide (24). RNA Scission by 5'-(5-Glycinamido-l,lO-phenanthro1ine)-ACTGCCT (OP-ACTGCCT). Two procedures were used to explore the scission of the RNA by the heptamer. Both gave equivalent results. Condition A a 10-pL solution of lac RNA (0.25 pM) and OP-heptamer (10 pM) in 50 mM Tris, pH 8.0/50 mM NaCl was incubated for 1h. The cleavage reaction was then initiated by adding 1pL of CuSO4 (20 pM) and 1pL of MPA (58 mM) at 4 "C. After overnight at 4 "C, the reaction was quenched Condition B: lac with 2,9-dimethyl-l,lO-phenanthroline. RNA (0.25pM) and OP-heptamer (10 pM) were mixed in 20 pL of buffer containing 25 mM of Tris-C1 (pH 6.51, NaCl(70 mM), MgC12 (20 mM), and 0.4mM of spermine at either 4 "C or room temperature. The cleavagereaction was initiated as described above and carried out at either 4 "C or room temperature. Quenching, Precipitation, and Electrophoresis. Cleavage reactions were quenched by the addition of 2,9dimethyl-0P to a final concentration of 1 mM. The reactions were precipitated by the addition of 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol for 2 h at -80 "C. The reactions were centrifuged, and the pellet was washed with 60 pL of 70% ethanol. The reactions were again centrifuged and evaporated to dryness. The pellet was redissolved in 10 pL of urea dyes (10 M urea, 1mM EDTA, 0.1 7% xylene cyanol, and 0.1 % bromophenol blue), heated at 90 "C for 3 min, and subjected to polyacrylamide gel electrophoresis on a 10% denaturing gel. The gel was run at 50 W for 2.5 h. Autoradiography was performed overnight at -20 "C. RNase H Cleavage of RNA-DNA Hybrids. RNA (50 000 dpm) was dissolved in the same buffer as above. After the heptamer was added to a final concentration of 4 pM, hybridization was allowed to proceed as before a t 22, 30, or 37 "C. Dithiothreitol was added to a final concentration of 0.1 mM. Ribonuclease H (2 units) was then added and the reaction was incubated for 20 min. The reaction was stopped by the addition of EDTA to a final concentration of 30 mM. The reactions were then precipitated and electrophoresed as described in the previous section.
RESULTS AND DISCUSSION Specificity of DNA Scission by HOP-Cu. Hoechst dye 33258 is a structurally constrained ligand and, with its crescent shape, preorganized to bind to theminor groove (17-19). Given the minor groove reactivity of 1,lOphenanthroline-copper, a very efficient chemical nuclease activity might be expected by conjugation to Hoechst 33258. In addition to the configuration of the dye, three factors should contribute to the high-affinity binding of HOP to the minor groove of DNA. First, Hoechst 33258 binds tightly to A-T-rich regions of the minor groove; second, OP-Cu itself binds preferentially to the minor groove (30);finally, quaternization of the piperazine adds additional positive charge to HOP. Two DNA sequences, the E. coli lac UV-5promoter/ operator and the transcription termination region of the mouse immunoglobulin pm gene, were used to assay the sequence specificity of the chemical nuclease activity of HOP. Both the 5'-labeled template strand and 3'4abeled nontemplate strand of the lac UV-5fragment were used to monitor the cleavage chemistry. Histograms summarizing the cleavage sites for both HOP-Cu and the unsubstituted OP-Cu for both strands of lac UV-5are presented in Figure la,b. Hoechst 33258 modifies the
72
Chen et al.
Bioconjugate Chem., Vol. 4, No. 1, 1993
S A A T T G T G A G C G G A T A A C A A T T T C A C A C 3 1 5 IO 15 20 25 S T T A A C A C T C G C C T A T T G T T A A A G T G G S
4 1 6 -
-
I
“I‘
8 -
-
IO
S A A T T G T G A G C G G A T A A C A A T T T C A C A C 3 1 5 10 15 20 25 3 T T A A C A C T C G C C T A T T G T T A A A G T G T G S
0
2
4
6
1
II I
Figure 1. Scission pattern of lac promoter/operator by OP-Cu (a, top) and by HOP-Cu (b, bottom). In lb, the stippled lines indicate the binding site of Hoechst 33258 determined using OPCu footprinting.
cleavage specificity of 1,lO-phenanthroline-copper.The sites of HOP scission correspond to a strong binding site of Hoechst 33258 on both strands that has been identified using OP-Cu footprinting. Specifically, the strong sites of cleavage evident at positions 13-15 on the lower (template) strand and 16-20 on the upper (nontemplate) strand are associated with a binding site indicated by the stippled bar. The scission sites of HOP-Cu evident from 1-4 appear to be associated with a binding site that is visualized primarily on the lower strand using OP-Cu also represented by stippled bars. In contrast to OP-Cu, where 5 pM ligand and 1.2 pM metal ion were used, only 0.01p M HOP-Cu2+was required for scission. Both sets of cleavage sites exhibit a 3’ stagger indicative of minor-groovescission (31). The sequence GTGTG extending from 23 to 27 is effectively cut by OP-Cu but not by HOP-Cu because it lacks runs of A-T. Generally, the results with the lac fragment indicate that, to a first approximation, the conjugation of Hoechst 33258 to phenanthroline restricts the specificity of the phenanthroline scission and concentrates it adjacent to A-T-rich regions. However, the specificity of HOP-Cu for these regions is clearly not stringent. The reason for the asymmetric binding and reactivity of Hoechst and HOP-Cu at the upstream binding site is not apparent. Both HOP-Cu and OP-Cu cut at all nucleotides as would be expected from deoxyribose-based chemistry. The second DNA fragment that was used as a substrate for HOP-Cu was derived from the region of transcription termination of the pm gene of the mouse IgM locus. This A-T-rich DNA fragment contains the binding site for a nuclear protein that may facilitate termination as well as the polyadenylation signal, AAATAAA, and the site of polyadenylation (26). Comparison of the scission pattern of the nontemplate strand by HOP-Cu and OP-Cu again indicates that the primary sequence specificity of the two reagents differ (Figure 2a,b). With this fragment, there is preferential cutting near A-T-rich regions, but this sequence preference is relaxed. The increased hydrophobicity and positive charge resultingfrom the alkylation of Hoechst 33258 by 5-(iodoacetamido)-l,l0-phenanthroline may increase the nonspecific binding affinity of HOPCu and diminish the bias for A-T-rich regions that would be expected solelyon the basis of the specificity of Hoechst 33258. Conjugating 1,lO-phenanthroline to other DNAbinding drugs is likely to lead to parallel results, especially if they are minor-groove ligands. Although the increased affinity of the chimeras diminish their specificity, it may make them useful intracellular footprinting reagents. Specificity of Scission by a 1,lO-PhenanthrolineLinked Helix-Turn-Helix Peptide. The E. coli Trp repressor has been converted into a site-specific nuclease by chemicallymodifying all the lysyl residues of the protein with iminothiolane and then alkylating the resulting thio groups with 5-(iodoacetamido)-l,l0-phenanthroline(11). This derivatized protein cleaved both the aroH and trpEDCBA operators. This experiment raised the interesting possibility that the helix-turn-helix domain of this protein alone might also be capable of targeting the nuclease activity of 1,lO-phenanthroline-copper. To investigate this question, P-2was synthesized. The sequence of P-2 is presented below along with that of the helixturn-helix domain of the wild-type repressor (P-1). The sequence of P-2 differs from that of P-1in two amino acid residues. To conform with the sequence of a mutant Trp superrepressor (321,a valine residue has been substituted for an alanine residue at sequence position 77 of the loop
BioconjugSe Chem., Val. 4, No. 1, 1993 73
Nuclease ActMty of 1.10-Phenanthrollne-Capper
HOP
iL
v-
u" w
OP
5'
5'
C
A
;: ;E-
&
::: =A-
-240-
..
-9an""
t-
!e
1
-220-
C t 1
G
, - [E
t G
-210-
C
. ,
4214 T
C A
3' 0
G
2
4
6
8
10
3.0
2
4
6
8
10
0
a b C d Figure 2. Scission pattern of p m transcription termination. (a, left) Lane a, control; lane b, G + A sequencing lane; lane c, scission by 0.01 p M HOP-Cu; lane d, scission by OP-Cu ([OP] = 5 pM, [CUI = 1.2 pM). Reaction was carried out in 50 mM Tris-Ac (pH 8.0),10 mM MgC12 and 100 mM KCl for 1.5 h at 37 "C for OP-Cu and for 20 h at 37 "C for HOP-Cu. (b, right) Densitometric scan of lanes c and d of panel 2a. For additional sequence information see Law et al. (26). region. In addition, a tryptophan has replaced a glycine residue at position 85 because structural studies have shown that the corepressor tryptophan binds adjacent to this glycine and possibly contributes to the stability of the helix-turn-helix format (33-36). An N- [14C]acetylcysteinyl residue has been added at the N-terminus of P-2 to provide a convenient site of attachment of 5-(iodoacetamido)-1,lO-phenanthroline.In contrast to the rigid preorganized structure of Hoechst 33258, the helix-turn-helix peptide (P-2) derived from the E. coli Trp repressor is a flexible, random coil in solution. Circular dichroism studies demonstrated that P-2 lacked a stable secondary structure. P-2
77 0P-N-[14C]acetyl-CysCl~ArgGlu-Leu-Lys-Asn-Glu-Leu-Gly-~-Gly-Ile85
-
Ala-Thr-Ile-Thr-Arg-Trp-Ser-Asn-CONH:! P-1
77 GIn-Arg-Glu-Leu-Lys-Asn-Glu-Leu-G~-~Gly-Ile-Ala-Thr-Ile-Thr-Arg85 Gly-Ser-Asn-CONH2
-
DNase I footprinting (37)fails to detect sequencespecific binding of P-2 to the aroH operator at the concentrations where the native protein gives a clear footprint. The DNase footprints are presented in Figure 3a (lanes a-d). These experiments, in agreement with circular dichroism studies, indicate that the helix-turnhelix conformationalformat evident in the native protein is unstable in solution but does not exclude the possibility that selective scission might be obtained if sequencespecific contacts between the operator DNA and peptide transitorily stabilizeP-2 in a helix-turn-helix format. This possibility is incorporated in the reaction scheme below
in which P-2 exists free in solution as a random coil but can also exist in a helix-turn-helix format characteristic of the native protein when bound to DNA (Scheme I). When P-2 was used to cleave aroH, its pattern of scission was dramatically different from that obtained with the chemically modified wild-type E. coli Trp repressor. Instead of a restricted number of discrete sites that were observed with the Trp repressor, multiple products were obtained in the cleavage of aroH by the peptide. As a control for the scission of aroH by P-2, the identical DNA fragment was cleaved by OP-Cu and the products separated simultaneously on the same sequencing gel. Analysis of the scission products of aroH produced by P-2 and OP-Cu by densitometry indicated that a different product distribution was generated, exactly where the products of the protein targeted scission migrated. In the other regions, the product distribution of the P-2 and OP-Cu scission were equivalent. Two parallel reactions are responsible for the product distribution observed with P-2. One is nonspecific and can be attributed to the background scission of the labeled DNA substrate by OP-Cu substituted with a nonspecific 5 substituent (i.e. a phenyl group or a random peptide) as in the kinetic mechanism summarized in eq 1. This reaction cannot be quenched by carrier DNA or RNA traps in solution because this cleavage reaction involves the binding of the OP-Cu complexes to the DNA. Since the cleavage pattern of OP-Cu is not sensitive to substituents at the 5-position lacking a specific affinity for DNA (3), this background scission is similar to that observed with OP-Cu. The second reaction which leads to the same products as that observed with the 1,lOphenanthroline-derivatized wild-type repressor must be due to the reaction of the peptide stabilized in the helixturn-helix format on the surface of the DNA. The complete kinetic scheme for the scission of aroH by P-2 is summarized in Scheme 11. The most important con-
74
Chen et al.
Biocm~t@€it8 Chem., VOl. 4, No. 1, 1993
Scheme I
a b c d e f a h i
DNase1 OP-trp rep (2pM) P-2(pM)
’+ +
t 0
- -
+ + + + 0
4- t t ’ C U + + / M P A
o - - -
250 50 250 50 -
-
+
- -
e
-
mndom conformation
helix-tum-helixconformation
OP-trprep(2pM) P - 2 (pM)
+
OP
.55
so.
.50 48
. 42 .
Scheme I1
4 46
46
- fl
38 40
36
31 26 -
34
34
32
P O
-
30 28 26
It-.
24
22
-
helm-tum-helix conformation
random conformation
42
22
-
-:
bP
=
background scission
ml
70
75
Gln - Arg -Glu-Leu- Lys -Asn -Glu- Leu- Gly -ValGly- Ile-Ala-Thr- Ile -Thr-Arg-Trp-Ser-Asn 80
85
A
OP-cu I
n
-cu
Distance +
Figure 3. (a, top) Binding of TrpR-derived peptide to aroH operator measured by targeted scission and DNase I footprinting. All reactions were carried out using 3’-labeled restriction fragment containing the aroH operator (1nM), 10 mM L-tryptophan in a Tris-HC1 buffer (30 mM, pH 8.0) with 100 mM KCl and 3 mM MgC12 at 25 “C. Reaction volumes were 20 pL. DNase I footprinting was carried out for 5 min at room temperature using 0.04 units of enzyme. The nuclease activity of OP-Cu and P-2 was activated by adding 1p L of CuS04 and 1 p L of MPA (58 mM) and then incubated for 2 h at 37 “C. Reactions were quenched by the addition of 2,9-dimethyl-l,lO-phenanthroline. (b, bottom) Panels A, B, and C are densitometric scans of lanes f, h, and i, respectively.
clusion of our studies using the helix-turn-helix peptide domain to target cleavage is that the binding energy available in sequence-specific contacts between DNA and peptide can stabilize a peptide in a functional conformation characteristic of the parent protein. This, however, does not exclude the stabilization of the helix-turnhelix conformation by other domains of the proteins, as has been recently suggested by Carey and her colleagues (38). Site-Specific Scission by 5’-OP-ATCCGCT-3’. Previous work has demonstrated that deoxyoligonucleotides linked at the 5’ end to 1,lO-phenanthroline can nick the target RNA following hybridization when the chemical nuclease is activated by addition of thiol and hydrogen peroxide; they can also direct the scission of the complementary sequence by RNase H (24). In those studies, the RNA (the same lac fragment used in the present studies; see Figure 4a), was heat denatured and reannealed in the presence of the scission reagent. In the present case, 5’OP-ATCCGCT-3’, the heptamer, was simply mixed with the lac RNA without disrupting its structure or causing strand displacement as would be the case following denaturation. The 5’-OP-ATCCGCT-3’-directed RNase H hydrolysis of the loop confirms the formation of the heteroduplex. However, activation of the chemical nuclease activity by addition of Cu2+and 3-mercaptopropionic acid not only cleaves the RNA at sequence position 13-15 but also at positions 3-5 (Figure 4b). Although positions 13-15 are adjacent in the linear sequence to the complementary sequence of ATCCGCT, positions 3-5 are not. Nicking of the phosphodiester backbone at these positions can be attributed to the proximity in space of the ribose moieties to the OP at the 5’ end of the hybridized heptamer. If single-stranded M-13 DNA is used as the substrate, scission is not observed at sequencepositions 3-5 but only adjacent to the complementary sequence (e.g. sequence positions 13-15). This result is consistent with the lack of stability of stem-loop structures of DNA. The scission efficiency of 5’-OP-ATmeCmeCGmeCT-3’ with the lac RNA is indistinguishable from that of 5’-OP-ATCCGCT-3’.
Nuclease AclM3, of 1.10-PhenanthrolIne-Capper
- 10 G
Bioconlugafe chem., Vol. 4, No. 1, 1993 75 + 10
C AUM +I GAGC CA GGUC GCU GU UGUGUGGAAUUGU GU CCAG CGA CA ACACACUUUAACA G A UAU - AAGG +20 AUAG -20
--G
- 40
IAGA
GA GCUCCCAGGCUC~
Ll-17
- 30
+ +1
c
+IO
A
ucu
+20
GGGU UCUCYGGUUAG CCAGA
‘u
+30
GAGC CCCA-AGGGAYCAAUC-GGUCY--- C U C G A +SO
+40
G G
G
HTV TAR
u
3 5: 7-
+20
911 I3
15
-
GGGU
UCUCYGGUUAG C ? ~ ~ G A G C U C C C A G G C U C ~ CCCA - AGGGAYCAAUC -GGUCYCUCGAGGGUCCGAG-J ~
+SO
/*;40
+30
scission Site
L1-17-TAR Pseudo-half-knot Figure 5. Probing the conformationof a pseudo-half-knotusing an OP-Cu-linked RNA (42).
guanosine binding site of the Tetrahymena ribozymes using an EDTA derivative of this nucleoside is another example of the use of chemical nucleases in probing the conformation of an RNA (43). CONCLUSIONS
f
2
3
Figure 4. Scission of a single-stranded RNA loop by OP-linked heptamer. (a, top) Secondary structure of lac RNA used as substrate. (b, bottom) Scission of lac RNA fragment by 5’-0PATCCGCT-3’. 5’-OP-ATCCGCT-3’(10p M ) was incubatedwith 0.25 pM lac RNA in 10 p L of 50 mM Tris, pH 8.0/50 mM NaCl for 1 hat 4 “C. The scission reaction was then initiatedby adding 1 pLof CuSOd (20pM) and 1 p L of MPA (58mM). After overnight reaction, the products were analyzed on a sequencing gel.
In effect, conjugates composed of OP and short deoxyoligonucleotides complementary to single-strandedRNA loops can act as a cross-linking reagent and assist in defining the three-dimensional conformation of RNAs. In this application, they are unique. Although OP-linked deoxyoligonucleotides can also be used for the identification of single-strandedloops, this structural information can alternativelybe obtained using base-specific chemical reagents (39), deoxyoligonucleotide-directed RNase H scission (40),or the deoxyoligonucleotide suppression of OP-Cu scission of single-stranded loops (41). A novel use of an OP-linked RNA to probe the threedimensional structure of the HIV TAR hairpin loop has recently been reported (42). In this experiment (Figure 5), the OP-linked ribonucleotide (Ll-17) was hybridized with the HIV TAR hairpin loop. Scission sites at C-19 and U-40provide evidence for the formation of the L117-HIV TAR complex presented in Figure 5. Although cleavage at U-40is predictable from the primary sequence of the two RNAs, scission at C-19 is not and therefore is supportive of the postulated complex. Mapping the
The goal of these studies has been to identifynew classes of targeting ligands which could modify or redirect the nuclease activity of 1,lO-phenanthrolinepper. On the basis of the independence of the specificity on the experimental ratio of copper ion to the 1,lO-phenanthroline-derivatizedcarrier,the reactive complex must consist of one copper per phenanthroline. In all previous cases of the targeting of the nuclease activity of 1,lO-phenanthroline-copper (11,12,14,16,24), a second phenanthroline is not necessary when affinity for nucleic acid is provided by the carrier. The conclusions of these studies can be summarized as follows. (1) HOP-Cu has proven to be an efficient scission reagent with different scission specificity than OP-Cu, but its specificity is not rigidly directed to A-T-rich regions. Hoechst dye 33258 is the first abiological functionality linked to the 5-position of phenanthroline which can alter the specificity of the chemical nuclease. Generally, 1,10-phenanthroline may diminish the binding specificity of an organic ligand (e.g. Hoechst dye 33258) upon conjugation because it will increasethe nonspecificbinding affinity of the chimera. However, the new reagent may have novel and useful new specificity. HOP-Cu, because of its high binding affinity, may be useful in the analysis of chromatin structure given the competition of Hoechst 33258 with high mobility group (HMG I) nonhistone chromosomal proteins (44). (2) P-2 nicks the aroH operator in the same sequence region as the protein conjugate. These results indicate that the binding energy of the DNA-peptide interaction can stabilize the peptide in the helix-turn-helix confor-
76 Bloconlugete Chem., Vol. 4, No. 1, 1993
mational format. However, significantcleavage is observed at flanking sequences because of the intrinsic affinity of 1,lO-phenanthrolinecopperfor t h e minor groove of DNA. Therefore, conjugating peptides derived from DNA binding domains to OP-Cu will not generate efficient and specific scission reagents if t h e peptides exist as random coils. Other studies of peptideDNA interactions using circular dichroism, tryptic digestion, and infrared spectroscopy have demonstrated the ability of DNA binding to stabilize the structure of a peptide (45-47). This is the first study to demonstrate this phenomenon at the level of nucleotide resolution. (3) Of t h e three targeting systems studied, only the deoxyoligonucleotide was capable of overriding the intrinsic specificity of 1,lO-phenanthroline-copper in its reaction with RNA. Nucleotidesadjacent in space,as well in sequence, t o single-stranded loops in RNA can be identified using short complementary deoxyoligonucleotides linked to OP-Cu. These reagents therefore are useful in mapping the 3-D structure of RNA. However, the scission may not be sufficiently robust to enhance the efficiency of these deoxyoligonucleotides as antisense reagents. ACKNOWLEDGMENT
This research was supported by USPHS GM 21199 and Office of Naval Research G r a n t N00014-86-K5024. W e acknowledge useful conversations with Theodore B. Thederahn and the assistance of E v a Chun in the preparation of this manuscript. LITERATURE CITED (1) Sigman, D. S. (1990) Chemical Nucleases. Biochemistry 29,
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