- Email: [email protected]
Targeting the minor groove of DNA
355
T a r g e t i n g t h e m i n o r g r o o v e of D N A David E Wemmer* and Peter B Dervan¢ Small molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome will be useful tools in molecular biology and, potentially, in human medicine. Pairing rules have been developed to control rationally the sequence specifity of minor groove binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Using simple molecular shapes and a two-letter aromatic amino acid code, pyrrole-imidazole polyamides achieve affinities and specificities comparable to DNA-binding proteins.
Addresses *Department of Chemistry, MC-1460, University of California, Berkeley, CA 94620-1460, USA; e-mail: [email protected] tDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA; e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:355-361
Figure 1
/
(a)
o NJ~ H2N+~-'~
/
H H
N-~
/NH2 ""~(+ NH2
(b)
/ ( ~
,o.2.J (c)
N
~
N
,
N
~
N.,---,~
"
/ o \_>_N
© Current Biology Ltd ISSN 0959-440X Abbreviations Im imidazole Py pyrrole H
I N °
/
,o -5(NH2
(d)
Introduction
Viewed from a distance, the regular helical grooves of DNA offer no clues about how proteins and small molecule ligands can recognize specific sequences. When viewed at the individual base-pair level, however, it is apparent that the edges of the base pairs offer arrays of hydrogen-bond donors and acceptors that have details determined by the DNA sequence. Studies by X-ray diffraction and N M R have shown that the local structure of DNA is sequence dependent. Long ago, it was recognized that there are more functional groups within the major groove [1], and hence it was surmised that most proteins would recognize that groove. This has been born out, however, proteins have also been found that recognize DNA from the minor groove [2,3] or that have tails that wrap into this groove and contribute to specificity [4-6]. A variety of small molecule ligands have also been fovnd to bind within the minor groove [7]. In this reviex~, we discuss the basis for sequence preference in the minor-groove binding of small ligands, and, using this basis, how ligands can be designed to target predetermined DNA. aromatic
°
2HN
http://biomednet.com/elecref/O959440XO0700355
Curved
I N
compounds
A wide variety of compounds have been discovered or synthesized that bind preferentially bind in the minor groove at A.T sequences. T h e s e compounds have several common features: they are comprised of aromatic rings;
© © 1997 Current Opinion in Struelural Biology
The structures of four A.T specific minor-groove ligands. (a) Netropsin. (b) Hoescht 33258. (c) Distamycin. (d) SN 6999.
they contain linkages such that they have an overall curvature that matches that of the floor of the minor groove; they have hydrogen-bond donors on the inside edge; and they have one or more positive charges. Distamycin and netropsin are examples of natural products in this catego~; and Hoescht 33258 and SN-6999 are examples of synthetic ones (Fig. 1). Early studies showed that binding of distamvcin to the simple polymers polvdA.polvdT and polyd(A.T) occurred with similar affinity [8]; however, recent quantative footprinting work examining a wide variety of specific sites with A.T pairs within longer DNA indicates that some A,T combinations have much higher affinity than others [9]. In particular, those that have all the A residues on one strand tend to have the highest affinity, A to T steps decrease affinity only slightly, but T to A steps lower affinity more substantially.
356
Nucleic acids
Figure 2
(a)
(b)
~c 1997 CurrentOpinionin StructuralBiology Structures derived from NMR are shown for (a) the 2:1 distamycin complex with AAATF, and (b) the 1:1 distamycin complex with AATT. Distamycin is highlighted in dark gray. The much narrower minor groove of the 1:1 complex is apparent in (b).
Crystallographic and NMR studies have shown that such tight binding sequences have an unusually narrow minor groove [10-12]. The structures of DNA-ligand complexes with these sequences have indicated that the very narrow groove is maintained within the complex [13,14] and that there is a tight fit of ligand in the groove, which suggests particularly good van der Waals contacts between the surface of the ligand and the walls of the groove. In addition to the good fit at these sites, hydrogen bonds occur between the ligand and the base-pair edges. The positively charged groups on the ligand lie deep within the groove where the electrostatic potential is high.
Groove flexibility and 2:1 binding NMR studies of distamycin complexes with DNA oligomers containing five or more A-T pairs indicated a new form of complex, which has been shown to have two distamycin molecules bound antiparallel, side-by-side in the groove (Fig. 2) [15]. This clearly demonstrates the flexibility of the minor groovc, as the groove width has increased by 3.5-4.& in going from 1:1 to 2:1 binding. Titration calorimetry studies [16] have further shown that the first ligand has an association constant of about 107 M -1 and the second ligand one of about 106M -1 on an AAATT site. For the sequence AAAAA, it has been found that the 2:1 binding mode is disfavored relative to 1:1 ( K 2 / K t >0.01). The alternating sequence TATAT, however, shows exactly the opposite behavior--the 2:1 mode is favored over the
1:1 ( K z / K 1 > 100) [17]. While the ratio changes dramatically (by 104), it appears that the product K 2 . K 1 remains about constant (which reflects that the total binding free energy remains constant). Substitutions of I-C for A-T, which leave the minor groove functional groups unchanged, also enhance the 2:1 mode relative to the 1:1 mode. In fact, the only crystal structure of a DNA with distamycin bound in the 2:1 motif is that of an (IC) 4 oligomer [18]. These data indicate that DNA groove shape and flexibility strongly affect the binding behavior. It is worth noting that, to date, distamycin is the only member of the family of compounds that has been seen to bind in the 2:1 mode. As netropsin has positive charges on both ends, the 2:1 binding mode would be disfavored electrostatically for this ligand.
Rational design -targeting specific sequences It is natural to ask whether any of these ligand structures can be rationally modified to alter their sequence specificity. From a knowledge of the structure of the 1:1 netropsin.AATT complex, Kopka et al. [19] and Lown et a]. [20] suggested that converting one of the pyrrolc (Py) rings to imidazolc (Im) should change the specificity for the contacted pair from A.T to G-C. When such analogs of netropsin were made, however, they were found to have lost preference for A.T sites, but they did not show the expected preference for G.C. In retrospect, choosing netropsin as a model with its positive charges on both ends of the ligand probably restricted these molecules to
Targeting the minor groove of DNA Wemmer and Dervan
357
Figure 3
(a)
~
(CH2)4~
~k I
"
(c)
(b)
~ ~~~.= o ~, ,U'~-~'--~"
14--"
o
...N.7~
-->.'--
,'~N---/ ......
/
'~
/ /
•
5'
y
HN '~ l
,
)
dH..........
......./
....
.... '>N. --- HN-%
7
.Ny--N 0 5'
°
.......~ - ~ . . . H N ~ O
o~ ~ o/~>/. /
,
::.
5'
3' 1997 Current Opinion in Structural Biology
Structures of linked polyamide ligands bound to the same target DNA. (a) The H-pin: ImPyPy-C4-AcPyPyPy.5'-TGTTA-3' [30]. (b) The hairpin: ImPyPy-T-PyPyPy.5"-TGI-FA-3' [32]. (¢) The cyclic ligand: cyclo-(ImPyPy-T-PyPyPy-'~).5'-TGTTA-3' [35]. DNA bases are represented by circles. Hydrogen bonds are shown as dotted lines.
Figure 4
(a)
(b)
¢: 1997 Current Opinion in Structural Biology
The structure of the hairpin motif bound to a target DNA, (a) View from the front of the DNA. (b) View into the minor groove of the DNA. The positioning of the linker deep within the groove is apparent.
binding as 1:1 complexes. It was soon demonstrated that changing Py to Im is insufficient for targeting G.C base pairs [21-231. Instead, a binary code of two rings (Im/Py) is required to distinguish G.C and C.G from A-T [21-23].
A three-ring polyamide, ImPyP'~, has been synthesized and shown, using footprinting studies, to have a marked preference for five base-pair sequences containing not one, but two G.C base pairs [21]. T h e fact that the two G-C
358
Nucleicacids
pairs are targeted in the second and fourth positions of W G W C W sequences (where W represents either A.T or T.A) suggests that these ligands can only be binding as an antiparallel dimer wherein Im]Py targets G.C and Py/Im targets C.G [21]. Structural studies have confirmed the 2:1 binding motif and that each G-C pair has an Im on the G strand and a Py on the C strand [22]. As a test for generality, one ring on the homodimer has been converted to a Py, and the new heterodimer, ImPyPy/PyPyPy, binds a new predicted sequence, WGWWW, revealing the potential of the new pairing rules [24]. To test how far these rules can be taken, the compound I m P y I m P y has been made and shown to bind to a core G C G C target site, as anticipated [25,26]. In all studies of distamycin, the alkyl chain has been observed to bind at an A.T site and therefore, as expected, the target sites are W G C G C W . T h e binding sites, specificity and 2:1 nature of the complex have been verified using both footprinting [26] and N M R [25]. T h e set of simple pairing rules for these polyamide ligands can be summarized: use side-by-side binding of pairs of Im and/or Py rings, one pair per targeted base pair; use Im for the G strand and Py for the C strand to target a G.C base-pair site; and use two Py rings to target A.T or T-A. To date, ligands designed using these rules have always bound the designated target sequence. Some testing of specificity has been done, and often a single 'mismatch' ( e . g . G . C in place of C.G) leads to a loss of a factor of ten or more in binding affinity, although there are some base combinations for which the discrimination is lower, and some for which it is higher [26]. There is also some variation in binding affinity for sequences containing different A.T or T-A combinations, although these are nominally degenerate in their design [27]. A complete understanding of what controls the degree of specificity has not yet been developed.
Linked systems Many years ago, distamycin analogs containing four or five Py rings were shown to maintain their A.T specificity and to have increased affinity [28]. Six or more rings, however, lead to a decrease in affinit'~: It has been suggested that the ligand and the D N A groove get 'out of register', either in length or curvature of the ligand. Introduction linkers that have torsional flexibility have also been found to allow longer ligands to be made, which retain high binding affinity and A.T specificity [29]. In the side-by-side binding mode described above, two ligands are required, and it is natural to think of using covalent linkage to reduce the entropy loss upon binding and to enhance the specificity by requiring that the linked units bind at the same site. Two approaches have been described, one bridging the ligands by connecting the N-methyl of the Pys (called H-pins) [30,31], and the other
linking polyamides head-to-tail (called hairpins; Fig. 3) [32-34]. Both lead to enhanced affinity and specificity. As is apparent from the structural models of the 2:1 complexes (Fig. 2), the linkers in H-pins protrude out from the groove and do not contact the D N A at all, whereas the hairpin linker is buried within the minor groove. Figure 5
AGATA
AGATA
AGATA
TCTAT
TCTAT
TCTAT
GTAGCGCTAG OOO(~ +
AGTACT
CATCGCGATC
TCATGA
CTGATATACAC
CTTTTAGACAAATTC
GACTATATGTG
GAAAATCTGTTTAAG
~~'
AGCCATTAGA TCGGTAATCT 1997 Current Opinion in StructuraJ Biology
Schematic diagrams of different types of polyamide-DNA complexes that have been characterized. Each imidazole (Im) is shown as a shaded circle, each pyrrole (Py) ring is shown as an open circle, and linkers and tails are indicated by black lines.
Different linear amino acid linkers have also been tested in making hairpins, varying from 1-4 methylene carbons in length (designated ~, [3, y, 5). With the ~ linker (glycine), the halves of the ligand can clearly not fold back to form the hairpin. Although folding back can occur with the ]3 linker, the turn is strained and is energetically unfavorable. T h e y-hairpin turn can form easily (having a geometry similar to half of a cyclohexane), and molecules with this linker have increased affinity over the unlinked dimcrs by two orders of magnitude [32]. When the 8 linker is used. the turn forms properly, but the affinity is lower that for the ylinker. N M R studies show that these linkers sit deep in the groove (Fig. 4), and steric conflict of the 8 chain with the walls of the groove seems to be responsible for the lowered affinity.
Targeting the minor groove of DNA Wemmer and Dervan
359
Figure 6 (a)
(b)
o
/NHCO2CH3
Ho'-F~ /
,,c
o
o I
I
.["~'~'0 °
HOo,
(-.-"-0~ . ~
NHCH2CH3
.
?Ha [I
OH OH 0 O" ~ H30" " ~ 0 . ~
CHa\ ~07 H
~'OCH3OH OCH3
.o 0o-,;-o-o-o-o-o-o-7
OH 1997
CurrentOpinionin StructuralBiology
Structures of two minor groove binding ligands. (a) Mithramycin. (b) Calicheamicin YI".
T h e limiting case for minimizing conformational flexibility is c l o s u r e - - m a k i n g a cyclic ligand using two y linkers. This has been done to make a g-ImPyPy-y-PyPyPy ligand (with one Py methyl extended to an alkylamine to enhance solubility) [35]. T h e affinity is indeed high, reaching over 109M -1. T h e sequence discrimination has been found to not be as good as the hairpin, however, suggesting that some flexibility is required to optimize local contacts. With the c~-linked and [3-linked ligands, it seems possible that binding can occur in an extended 2:1 mode. Remarkably, two binding modes have been identified [36"], one fully overlapping the two ligands in the groove, and the other 'slipped' to use the I m P y P y segments in a 2:1 motif, but the P y P y P y segments in a 1:1 mode. Structure of both types have been analyzed using footprinting and detailed N M R studies [37"]. T h e affinities are high, but not as high as hairpin ligands containing four or five rings in each half, which bind at subnanomolar concentrations [38"]. Hairpins that bind in the 2:1 mode and that have extensions with 1:1 binding have also been prepared [39°], and these bind with v e w high affinity. A summary of the observed binding modes is given in Figure 5.
are shown in Figure 6. Mithramycin and chromomycin (both very similar compounds) dimerize via a magnesium atom and then bind to the minor groove, which strongly distorts the local structure [41]. While many contacts can be seen that probably contribute to the specificity, it is not yet apparent how to modify the covalent structure to systematically change this specificity. T h e arylpolysaccharide from calicheamicin (an enediyne antibiotic) shows sequence preference in binding (preferring polypyrimidine tracts), and N M R studies of the complex have been carried out [42-44]. T h e relatively hydrophobic sugars make contacts with the walls of the groove, and both particular bonds (e.g. N - O linking two sugars) and particular atoms (e.g. aromatic iodine) probably contribute to the specificity. Although this has been touted as a new paradigm for general minor-groove recognition [45], the only concrete example to appear has been a dimer of this polysaccharide [46]. T h e greater complexity of these molecules makes discerning contributions to specificity difficult and also complicates preparation of variants.
Synthesis
Conclusions
T h e first polyamides were made in a labor-intense effort, which required step-wise solution chemistry that coupled Py or Im amino acids. No~, however, solid-phase synthesis of polyamides is possible [40"*]. T h e high coupling yields achieved makes possible the synthesis of analogs with more rings in larger quantities. Importantly. the synthetic effort spent per molecule has been reduced from months to a few days. This has been important advance for the exploration of the use of these synthetic ligands.
While the Py-Im polyamide approach has limitations (T.A versus A.T discrimination has yet to be achieved), these ligands have achieved many of the goals of rational sequence-specific ligand design. T h e y are able to bind with affinities and specificities for D N A comparable with naturally occurring DNA-binding proteins [38°*]. Unlike p r o t e i n - D N A recognition, the chemical principles elucidated are sufficiently simple that the method targets predetermined D N A sequences. Therefore, the next frontier may be the use of these synthetic ligands in biology and human medicine [47]. In very recent work, such ligands have been shown to be cell permeable and can inhibit the transcription of specific genes [47].
Generalization of other binding motifs Over the past few years, a number of structures for other minor-groove complexes have been s o l v e d - - t w o
360
Nucleic acids
References and recommended reading
20.
Lown JW, Krowicki K, Bhat UG, Skorobogaty A, Ward B, Dabrowiak JC: Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity. Biochemistry 1986, 25:7408-7416.
21.
Wade WE, Mrksich M, Dervan PB: Design of peptides that bind in the minor groove of DNA at 5"-(,AT)G(,AT)C(,AT)-3" sequences by a dimeric side-by-side motif. J Am Chem Soc 1992, 114:8783-8794.
22.
MrksichM, Wade WS, Dwyer TJ, Geierstanger BH, Wemmer DE, Dervan PB: Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin. Proc Nat/Acad Sci USA 1992, 89:7568-?590.
23.
Geierstanger BH, Dwyer TJ, Bathini Y, Lown JW, Wemmer DE: NMR characterization of a heterocomplex formed by distamycin and its analog 2-1mD with d(CGCAAGTTGGC):d(GCCAACTTGCG): preference for the 1:1:1 2-1mD:Dst:DNA complex over the 2:1 2-1mD:DNA and the 2:1 Dst:DNA complexes. J Am Chem Soc 1993, 115:4474-4482.
24.
MrksichM, Dervan PB: Antiparallel side-by-side heterodimer for sequence-specific recognition in the minor groove of DNA by a distamycin / 1-methylimidazole-2-carboxamide-netropsin pair. J Am Chem Soc 1993, 115:2572-2576.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • o of outstanding interest 1.
Seeman NC, Rosenberg JM, Rich A: Sequence-specific recognition of double helical nucleic acids by proteins. Proc Nat/Acad Sci USA 1976, 73:804-808.
2.
Nikolov DB, Hu S-H, Lin J, Gasch A, Hoffman A, Horikoshi M, Chua N-H, Roeder RG, Burley SK: Crystal structure of TFIID TATA-box binding protein. Nature 1992, 360:40-46.
3.
Werner MH, Huth JR, Bronenborn AM, Clore GM: Molecular basis of human 46X,Y sex reversal revealed from the threedimensional solution structure of the human S R Y - D N A complex. Cell 1995, 81:705-714.
4.
Billeter M, Qian YQ, Otting G, MLiller M, Gehring W, WSrich K: Determination of the NMR solution structure of an Antennapedia homeodomain-DNA complex. J Mo/ Bio/1993, 234:1084-1097.
5.
Feng J-A, Johnson RC, Dickerson RE: Hin recombinase bound
to DNA: the origin of specificity in major and minor groove interactions. Science 1994, 263:348-355. 6.
Schumacher MA, Choi KY, Zalkin H, Brennan RG: Crystal structure of Lacl member, PurR, bound to DNA: minor groove binding by c¢ helices. Science 1994, 266:763-770.
25.
Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Design of a G.C-specific DNA minor groove-binding peptide. Science 1994, 266:646-650.
7.
Geierstanger BH, Wemmer DE: Complexes of the minor groove of DNA. Annu Rev Biophys Biomo/ Struct 1995, 24:463-493.
26.
8.
Breslauer KJ, Remata DP, Chou W-Y, Ferrante R, Curry J, Zaunczkowski D, Snyder JG, Marky LA: Enthalpy-entropy compensations in drug-DNA binding studies. Proc Nat/Acad Sci USA 1987, 84:8922-8926.
MrksichM, Dervan PB: Recognition in the minor groove of DNA at 5'-(,AT)GCGC(,AT)-3' by a four ring tripeptide dimer. Reversal of the specificity of the natural product distamycin. J Am Chem Soc 1995, 117:3325-3332.
2?.
9.
Abu-Daya A, Brown PM, Fox KR: DNA sequence preferences of several AT-selective minor groove binding ligands. Nucleic Acids Res 1995, 23:3385-3392.
White S, Baird EE, Dervan PB: Effects of the A.T/T.A degeneracy of th pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 1996, 35:12532-1253Z
28.
10.
Drew HR, Wing RM, Takano T, Broka C, Tanaka S, Itakura K, Dickerson RE: Structure of a B-DNA dodecamer: conformation and dynamics. Proc Nat/Acad Sci USA 1981,78:2179-2183.
Youngquist RS, Dervan PB: Sequence-specific recognition of B-DNA by oligo(N-methylpyrrole-carboxamide)s. Proc Nat/Acad Sci USA 1955, 82:2565-2569.
29.
Youngquist RS, Dervan PB: A synthetic peptide binding 16 base pairs of ,AT double helical DNA. J Am Chem Soc 1987, 109:7564-7566.
11.
NelsonHCM, Finch JT, Luisi BR, Klug A: The structure of an oligo(dA).oligo(dT) tract and its biological implications. Nature 1987, 330:221-226.
30.
12.
Chuprina VB, Lipanov AA, Federoff OYu, Kim SG, Kintanar A, Reid BR: Sequence effects on local DNA topology. Proc Nat/ Acad Sci USA 198'7, 88:9087-9091.
MrksichM, Dervan PB: Design of a covalent peptide heterodimer for sequence-specific recognition in the minor groove of double helical DNA. J Am Chem Soc 1994, 116:3663-3664.
31.
Dwyer TJ, Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Structural analysis of covalent peptide dimers, bis(pyridine2-carboxamidonetropsin)(CH2)3_ 6 in complex with 5'-TGACT3" sites by two dimensional NMR. J Am Chem Soc 1993, 115:9900-9906.
13.
Kopka ML, Yoon C, Goodsell D, Pjura P, Dickerson RE: Binding of an antitumor drug to DNA. J Mo/Bio/1985, 183:553-563.
14.
Coil M, Frederick CA, Wang AH-J, Rich A: A bifurcated hydrogen-bonded conformation in the d(A.T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin. Proc Nat/Acad Sci USA 198"7, 84:8385-8389.
32.
Pelton JG, Wemmer DE: Structural characterization of a 2:1 distamycin A-d(CGCAAATTGGC) complex by two-dimensional NMR. Proc Nat/Acad Sci USA 1989, 86:5723-5?27.
MrksichM, Parks ME, Dervan PB: Hairpin peptide motif. A new class of oligopeptides for sequence-specific recognition in the minor groove of double-helical DNA. J Am Chem Soc 1994, 116:7983-7988.
33.
ParksME, Baird EE, Dervan PB: Optimization of the hairpin polyamide design for recognition of the minor groove of DNA. J A m Chem Soc 1996, 118:6147-6152.
34.
Swalley SE, Baird EE, Dervan PB: Recognition of a 5"-(A,T)GGG(,AT)2-3' sequence in the minor groove of DNA by an eight-ring hairpin polyamide. J Am Chem Soc 1996, 118:8198-8206.
35.
Cho J, Parks ME, Dervan PB: Cyclic polyamides for recognition in the minor groove of DNA. Proc Nat/Acad Sci 1995,
15.
16.
17.
RentzeperisD, Marky LA, Dwyer TJ, Geierstanger BH, Pelton JG, Wemmer DE: Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure. Biochemistry 1995, 34:2937-2945. WemmerDE, Geierstanger BH, Fagan PA, Dwyer TJ, Jacobsen JP, Pelton JG, Ball GE, Leheny AR, Chang WH, Bathini Y e t aL: Minor groove recognition of DNA by distamycin and its analogs. In Structural Biology: The State of the Art, Proceedings of the 8th Conversation. Edited by Sarma RH, Sarma MH. Albany, NY: Adenine Press; 1994:301-323.
18.
Chen X, Ramakrishnan B, Rao ST, Sundaralingam M: Binding of two Distamycin A molecules in the minor groove of an alternating B-DNA duplex. Nat Struct Biol 1994, 1:169-175.
19.
Kopka ML, Yoon C, Goodsell D, Pjura P, Dickerson RE: The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc Nat/Acad Sci USA 1985, 82:1376-1380.
92:10389-10392.
36.
TraugerJW, Baird EE, Mrksich M, Dervan PB: Extension of sequence-specific recognition in the minor groove of DNA by pyrrole-imidazole polyamides to 9-13 base pairs. J Am Chem Soc 1996, 118:6160-6166. This paper describes extended binding motifs that show that sequencespecific recognition can be extended to rather long target sequences. •
37. •
Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Extending the recognition site of designed minor groove binding molecules. Nat Struct Bio/1996, 3:321-324.
Targeting the minor groove of DNA Wemmer and Dervan
361
A structural characterization of the 'slipped' extended binding motif is presented, which shows the importance of linkers, and the good fit of ligand and DNA throughout the complex.
42.
Walker S, Murnick J, Kahne D: Structural characterization of a calicheamicin-DNA complex by NMR. J Am Chem Soc 1994, 115:7954-7961.
38. **
43.
Ikemoto N, Kumar RA, Ling l-r, Ellestad GA, Danishefsky SJ, Patel DJ: Calicheamicin-DNA complexes- warhead alignment and saccharide recognition of the minor groove. Proc Nat/Acad Sci USA 1995, 92:10506-10510.
44.
Paloma LG, Smith JA, Chazin WJ, Nicolaou KC: Interaction of calicheamicin with duplex D N A - r o l e of the oligosaccharide domain and identification of multiple binding modes. J Am Chem Soc 1994, 116:369?-3?08.
45.
KahneD: Strategies for the design of minor groove binders: a reevaluation based on the mergence of site-selective carbohydrate binders. Chem Bio/1995, 2:9-12.
46.
Bifulco G, Galeone A, Gomezpaloma L, Nicolaou KC, Chazin WJ: Solution structure of the head-to-head dimer of calicheamicin oligosaccharide domain and d(CGTAGGATATCCTACG)2. J Am Chem Soc 1996, 118:881 ?-8824.
47.
Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB: Regulation of gene expression by small molecules, Nature 1997, in press.
Trauger JW, Baird EE, Dervan PB: Recognition of DNA by designed ligands at subnanomolar concentrations. Nature 1996, 382:559-561. The authors show that four ring hairpin ligands bind with very high affinity and good specificity. 39.
Trauger JW, Baird EE, Dervan PB: Extended hairpin polyamide , motif for sequence-specific recognition in the minor groove of DNA. Chem Bio/1996, 3:369-377. This paper shows that one of the many possible extensions of the extended binding motif is functional, giving high affinity and specificity. 40. o-
Baird EE, Dervan PB: Solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J Am Chem Soc 1996, 118:6141-6146. This paper describes the synthetic enhancements that make practical the exploration of the binding motifs of polyamides and their applications. 41.
Gao X, Mirau P, Patel DJ: Structure refinement of the chromomycin dimer-DNA oligomer complex in solution. J Mo/ Bio/1992, 223:259-279.
T a r g e t i n g t h e m i n o r g r o o v e of D N A David E Wemmer* and Peter B Dervan¢ Small molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome will be useful tools in molecular biology and, potentially, in human medicine. Pairing rules have been developed to control rationally the sequence specifity of minor groove binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Using simple molecular shapes and a two-letter aromatic amino acid code, pyrrole-imidazole polyamides achieve affinities and specificities comparable to DNA-binding proteins.
Addresses *Department of Chemistry, MC-1460, University of California, Berkeley, CA 94620-1460, USA; e-mail: [email protected] tDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA; e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:355-361
Figure 1
/
(a)
o NJ~ H2N+~-'~
/
H H
N-~
/NH2 ""~(+ NH2
(b)
/ ( ~
,o.2.J (c)
N
~
N
,
N
~
N.,---,~
"
/ o \_>_N
© Current Biology Ltd ISSN 0959-440X Abbreviations Im imidazole Py pyrrole H
I N °
/
,o -5(NH2
(d)
Introduction
Viewed from a distance, the regular helical grooves of DNA offer no clues about how proteins and small molecule ligands can recognize specific sequences. When viewed at the individual base-pair level, however, it is apparent that the edges of the base pairs offer arrays of hydrogen-bond donors and acceptors that have details determined by the DNA sequence. Studies by X-ray diffraction and N M R have shown that the local structure of DNA is sequence dependent. Long ago, it was recognized that there are more functional groups within the major groove [1], and hence it was surmised that most proteins would recognize that groove. This has been born out, however, proteins have also been found that recognize DNA from the minor groove [2,3] or that have tails that wrap into this groove and contribute to specificity [4-6]. A variety of small molecule ligands have also been fovnd to bind within the minor groove [7]. In this reviex~, we discuss the basis for sequence preference in the minor-groove binding of small ligands, and, using this basis, how ligands can be designed to target predetermined DNA. aromatic
°
2HN
http://biomednet.com/elecref/O959440XO0700355
Curved
I N
compounds
A wide variety of compounds have been discovered or synthesized that bind preferentially bind in the minor groove at A.T sequences. T h e s e compounds have several common features: they are comprised of aromatic rings;
© © 1997 Current Opinion in Struelural Biology
The structures of four A.T specific minor-groove ligands. (a) Netropsin. (b) Hoescht 33258. (c) Distamycin. (d) SN 6999.
they contain linkages such that they have an overall curvature that matches that of the floor of the minor groove; they have hydrogen-bond donors on the inside edge; and they have one or more positive charges. Distamycin and netropsin are examples of natural products in this catego~; and Hoescht 33258 and SN-6999 are examples of synthetic ones (Fig. 1). Early studies showed that binding of distamvcin to the simple polymers polvdA.polvdT and polyd(A.T) occurred with similar affinity [8]; however, recent quantative footprinting work examining a wide variety of specific sites with A.T pairs within longer DNA indicates that some A,T combinations have much higher affinity than others [9]. In particular, those that have all the A residues on one strand tend to have the highest affinity, A to T steps decrease affinity only slightly, but T to A steps lower affinity more substantially.
356
Nucleic acids
Figure 2
(a)
(b)
~c 1997 CurrentOpinionin StructuralBiology Structures derived from NMR are shown for (a) the 2:1 distamycin complex with AAATF, and (b) the 1:1 distamycin complex with AATT. Distamycin is highlighted in dark gray. The much narrower minor groove of the 1:1 complex is apparent in (b).
Crystallographic and NMR studies have shown that such tight binding sequences have an unusually narrow minor groove [10-12]. The structures of DNA-ligand complexes with these sequences have indicated that the very narrow groove is maintained within the complex [13,14] and that there is a tight fit of ligand in the groove, which suggests particularly good van der Waals contacts between the surface of the ligand and the walls of the groove. In addition to the good fit at these sites, hydrogen bonds occur between the ligand and the base-pair edges. The positively charged groups on the ligand lie deep within the groove where the electrostatic potential is high.
Groove flexibility and 2:1 binding NMR studies of distamycin complexes with DNA oligomers containing five or more A-T pairs indicated a new form of complex, which has been shown to have two distamycin molecules bound antiparallel, side-by-side in the groove (Fig. 2) [15]. This clearly demonstrates the flexibility of the minor groovc, as the groove width has increased by 3.5-4.& in going from 1:1 to 2:1 binding. Titration calorimetry studies [16] have further shown that the first ligand has an association constant of about 107 M -1 and the second ligand one of about 106M -1 on an AAATT site. For the sequence AAAAA, it has been found that the 2:1 binding mode is disfavored relative to 1:1 ( K 2 / K t >0.01). The alternating sequence TATAT, however, shows exactly the opposite behavior--the 2:1 mode is favored over the
1:1 ( K z / K 1 > 100) [17]. While the ratio changes dramatically (by 104), it appears that the product K 2 . K 1 remains about constant (which reflects that the total binding free energy remains constant). Substitutions of I-C for A-T, which leave the minor groove functional groups unchanged, also enhance the 2:1 mode relative to the 1:1 mode. In fact, the only crystal structure of a DNA with distamycin bound in the 2:1 motif is that of an (IC) 4 oligomer [18]. These data indicate that DNA groove shape and flexibility strongly affect the binding behavior. It is worth noting that, to date, distamycin is the only member of the family of compounds that has been seen to bind in the 2:1 mode. As netropsin has positive charges on both ends, the 2:1 binding mode would be disfavored electrostatically for this ligand.
Rational design -targeting specific sequences It is natural to ask whether any of these ligand structures can be rationally modified to alter their sequence specificity. From a knowledge of the structure of the 1:1 netropsin.AATT complex, Kopka et al. [19] and Lown et a]. [20] suggested that converting one of the pyrrolc (Py) rings to imidazolc (Im) should change the specificity for the contacted pair from A.T to G-C. When such analogs of netropsin were made, however, they were found to have lost preference for A.T sites, but they did not show the expected preference for G.C. In retrospect, choosing netropsin as a model with its positive charges on both ends of the ligand probably restricted these molecules to
Targeting the minor groove of DNA Wemmer and Dervan
357
Figure 3
(a)
~
(CH2)4~
~k I
"
(c)
(b)
~ ~~~.= o ~, ,U'~-~'--~"
14--"
o
...N.7~
-->.'--
,'~N---/ ......
/
'~
/ /
•
5'
y
HN '~ l
,
)
dH..........
......./
....
.... '>N. --- HN-%
7
.Ny--N 0 5'
°
.......~ - ~ . . . H N ~ O
o~ ~ o/~>/. /
,
::.
5'
3' 1997 Current Opinion in Structural Biology
Structures of linked polyamide ligands bound to the same target DNA. (a) The H-pin: ImPyPy-C4-AcPyPyPy.5'-TGTTA-3' [30]. (b) The hairpin: ImPyPy-T-PyPyPy.5"-TGI-FA-3' [32]. (¢) The cyclic ligand: cyclo-(ImPyPy-T-PyPyPy-'~).5'-TGTTA-3' [35]. DNA bases are represented by circles. Hydrogen bonds are shown as dotted lines.
Figure 4
(a)
(b)
¢: 1997 Current Opinion in Structural Biology
The structure of the hairpin motif bound to a target DNA, (a) View from the front of the DNA. (b) View into the minor groove of the DNA. The positioning of the linker deep within the groove is apparent.
binding as 1:1 complexes. It was soon demonstrated that changing Py to Im is insufficient for targeting G.C base pairs [21-231. Instead, a binary code of two rings (Im/Py) is required to distinguish G.C and C.G from A-T [21-23].
A three-ring polyamide, ImPyP'~, has been synthesized and shown, using footprinting studies, to have a marked preference for five base-pair sequences containing not one, but two G.C base pairs [21]. T h e fact that the two G-C
358
Nucleicacids
pairs are targeted in the second and fourth positions of W G W C W sequences (where W represents either A.T or T.A) suggests that these ligands can only be binding as an antiparallel dimer wherein Im]Py targets G.C and Py/Im targets C.G [21]. Structural studies have confirmed the 2:1 binding motif and that each G-C pair has an Im on the G strand and a Py on the C strand [22]. As a test for generality, one ring on the homodimer has been converted to a Py, and the new heterodimer, ImPyPy/PyPyPy, binds a new predicted sequence, WGWWW, revealing the potential of the new pairing rules [24]. To test how far these rules can be taken, the compound I m P y I m P y has been made and shown to bind to a core G C G C target site, as anticipated [25,26]. In all studies of distamycin, the alkyl chain has been observed to bind at an A.T site and therefore, as expected, the target sites are W G C G C W . T h e binding sites, specificity and 2:1 nature of the complex have been verified using both footprinting [26] and N M R [25]. T h e set of simple pairing rules for these polyamide ligands can be summarized: use side-by-side binding of pairs of Im and/or Py rings, one pair per targeted base pair; use Im for the G strand and Py for the C strand to target a G.C base-pair site; and use two Py rings to target A.T or T-A. To date, ligands designed using these rules have always bound the designated target sequence. Some testing of specificity has been done, and often a single 'mismatch' ( e . g . G . C in place of C.G) leads to a loss of a factor of ten or more in binding affinity, although there are some base combinations for which the discrimination is lower, and some for which it is higher [26]. There is also some variation in binding affinity for sequences containing different A.T or T-A combinations, although these are nominally degenerate in their design [27]. A complete understanding of what controls the degree of specificity has not yet been developed.
Linked systems Many years ago, distamycin analogs containing four or five Py rings were shown to maintain their A.T specificity and to have increased affinity [28]. Six or more rings, however, lead to a decrease in affinit'~: It has been suggested that the ligand and the D N A groove get 'out of register', either in length or curvature of the ligand. Introduction linkers that have torsional flexibility have also been found to allow longer ligands to be made, which retain high binding affinity and A.T specificity [29]. In the side-by-side binding mode described above, two ligands are required, and it is natural to think of using covalent linkage to reduce the entropy loss upon binding and to enhance the specificity by requiring that the linked units bind at the same site. Two approaches have been described, one bridging the ligands by connecting the N-methyl of the Pys (called H-pins) [30,31], and the other
linking polyamides head-to-tail (called hairpins; Fig. 3) [32-34]. Both lead to enhanced affinity and specificity. As is apparent from the structural models of the 2:1 complexes (Fig. 2), the linkers in H-pins protrude out from the groove and do not contact the D N A at all, whereas the hairpin linker is buried within the minor groove. Figure 5
AGATA
AGATA
AGATA
TCTAT
TCTAT
TCTAT
GTAGCGCTAG OOO(~ +
AGTACT
CATCGCGATC
TCATGA
CTGATATACAC
CTTTTAGACAAATTC
GACTATATGTG
GAAAATCTGTTTAAG
~~'
AGCCATTAGA TCGGTAATCT 1997 Current Opinion in StructuraJ Biology
Schematic diagrams of different types of polyamide-DNA complexes that have been characterized. Each imidazole (Im) is shown as a shaded circle, each pyrrole (Py) ring is shown as an open circle, and linkers and tails are indicated by black lines.
Different linear amino acid linkers have also been tested in making hairpins, varying from 1-4 methylene carbons in length (designated ~, [3, y, 5). With the ~ linker (glycine), the halves of the ligand can clearly not fold back to form the hairpin. Although folding back can occur with the ]3 linker, the turn is strained and is energetically unfavorable. T h e y-hairpin turn can form easily (having a geometry similar to half of a cyclohexane), and molecules with this linker have increased affinity over the unlinked dimcrs by two orders of magnitude [32]. When the 8 linker is used. the turn forms properly, but the affinity is lower that for the ylinker. N M R studies show that these linkers sit deep in the groove (Fig. 4), and steric conflict of the 8 chain with the walls of the groove seems to be responsible for the lowered affinity.
Targeting the minor groove of DNA Wemmer and Dervan
359
Figure 6 (a)
(b)
o
/NHCO2CH3
Ho'-F~ /
,,c
o
o I
I
.["~'~'0 °
HOo,
(-.-"-0~ . ~
NHCH2CH3
.
?Ha [I
OH OH 0 O" ~ H30" " ~ 0 . ~
CHa\ ~07 H
~'OCH3OH OCH3
.o 0o-,;-o-o-o-o-o-o-7
OH 1997
CurrentOpinionin StructuralBiology
Structures of two minor groove binding ligands. (a) Mithramycin. (b) Calicheamicin YI".
T h e limiting case for minimizing conformational flexibility is c l o s u r e - - m a k i n g a cyclic ligand using two y linkers. This has been done to make a g-ImPyPy-y-PyPyPy ligand (with one Py methyl extended to an alkylamine to enhance solubility) [35]. T h e affinity is indeed high, reaching over 109M -1. T h e sequence discrimination has been found to not be as good as the hairpin, however, suggesting that some flexibility is required to optimize local contacts. With the c~-linked and [3-linked ligands, it seems possible that binding can occur in an extended 2:1 mode. Remarkably, two binding modes have been identified [36"], one fully overlapping the two ligands in the groove, and the other 'slipped' to use the I m P y P y segments in a 2:1 motif, but the P y P y P y segments in a 1:1 mode. Structure of both types have been analyzed using footprinting and detailed N M R studies [37"]. T h e affinities are high, but not as high as hairpin ligands containing four or five rings in each half, which bind at subnanomolar concentrations [38"]. Hairpins that bind in the 2:1 mode and that have extensions with 1:1 binding have also been prepared [39°], and these bind with v e w high affinity. A summary of the observed binding modes is given in Figure 5.
are shown in Figure 6. Mithramycin and chromomycin (both very similar compounds) dimerize via a magnesium atom and then bind to the minor groove, which strongly distorts the local structure [41]. While many contacts can be seen that probably contribute to the specificity, it is not yet apparent how to modify the covalent structure to systematically change this specificity. T h e arylpolysaccharide from calicheamicin (an enediyne antibiotic) shows sequence preference in binding (preferring polypyrimidine tracts), and N M R studies of the complex have been carried out [42-44]. T h e relatively hydrophobic sugars make contacts with the walls of the groove, and both particular bonds (e.g. N - O linking two sugars) and particular atoms (e.g. aromatic iodine) probably contribute to the specificity. Although this has been touted as a new paradigm for general minor-groove recognition [45], the only concrete example to appear has been a dimer of this polysaccharide [46]. T h e greater complexity of these molecules makes discerning contributions to specificity difficult and also complicates preparation of variants.
Synthesis
Conclusions
T h e first polyamides were made in a labor-intense effort, which required step-wise solution chemistry that coupled Py or Im amino acids. No~, however, solid-phase synthesis of polyamides is possible [40"*]. T h e high coupling yields achieved makes possible the synthesis of analogs with more rings in larger quantities. Importantly. the synthetic effort spent per molecule has been reduced from months to a few days. This has been important advance for the exploration of the use of these synthetic ligands.
While the Py-Im polyamide approach has limitations (T.A versus A.T discrimination has yet to be achieved), these ligands have achieved many of the goals of rational sequence-specific ligand design. T h e y are able to bind with affinities and specificities for D N A comparable with naturally occurring DNA-binding proteins [38°*]. Unlike p r o t e i n - D N A recognition, the chemical principles elucidated are sufficiently simple that the method targets predetermined D N A sequences. Therefore, the next frontier may be the use of these synthetic ligands in biology and human medicine [47]. In very recent work, such ligands have been shown to be cell permeable and can inhibit the transcription of specific genes [47].
Generalization of other binding motifs Over the past few years, a number of structures for other minor-groove complexes have been s o l v e d - - t w o
360
Nucleic acids
References and recommended reading
20.
Lown JW, Krowicki K, Bhat UG, Skorobogaty A, Ward B, Dabrowiak JC: Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity. Biochemistry 1986, 25:7408-7416.
21.
Wade WE, Mrksich M, Dervan PB: Design of peptides that bind in the minor groove of DNA at 5"-(,AT)G(,AT)C(,AT)-3" sequences by a dimeric side-by-side motif. J Am Chem Soc 1992, 114:8783-8794.
22.
MrksichM, Wade WS, Dwyer TJ, Geierstanger BH, Wemmer DE, Dervan PB: Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin. Proc Nat/Acad Sci USA 1992, 89:7568-?590.
23.
Geierstanger BH, Dwyer TJ, Bathini Y, Lown JW, Wemmer DE: NMR characterization of a heterocomplex formed by distamycin and its analog 2-1mD with d(CGCAAGTTGGC):d(GCCAACTTGCG): preference for the 1:1:1 2-1mD:Dst:DNA complex over the 2:1 2-1mD:DNA and the 2:1 Dst:DNA complexes. J Am Chem Soc 1993, 115:4474-4482.
24.
MrksichM, Dervan PB: Antiparallel side-by-side heterodimer for sequence-specific recognition in the minor groove of DNA by a distamycin / 1-methylimidazole-2-carboxamide-netropsin pair. J Am Chem Soc 1993, 115:2572-2576.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • o of outstanding interest 1.
Seeman NC, Rosenberg JM, Rich A: Sequence-specific recognition of double helical nucleic acids by proteins. Proc Nat/Acad Sci USA 1976, 73:804-808.
2.
Nikolov DB, Hu S-H, Lin J, Gasch A, Hoffman A, Horikoshi M, Chua N-H, Roeder RG, Burley SK: Crystal structure of TFIID TATA-box binding protein. Nature 1992, 360:40-46.
3.
Werner MH, Huth JR, Bronenborn AM, Clore GM: Molecular basis of human 46X,Y sex reversal revealed from the threedimensional solution structure of the human S R Y - D N A complex. Cell 1995, 81:705-714.
4.
Billeter M, Qian YQ, Otting G, MLiller M, Gehring W, WSrich K: Determination of the NMR solution structure of an Antennapedia homeodomain-DNA complex. J Mo/ Bio/1993, 234:1084-1097.
5.
Feng J-A, Johnson RC, Dickerson RE: Hin recombinase bound
to DNA: the origin of specificity in major and minor groove interactions. Science 1994, 263:348-355. 6.
Schumacher MA, Choi KY, Zalkin H, Brennan RG: Crystal structure of Lacl member, PurR, bound to DNA: minor groove binding by c¢ helices. Science 1994, 266:763-770.
25.
Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Design of a G.C-specific DNA minor groove-binding peptide. Science 1994, 266:646-650.
7.
Geierstanger BH, Wemmer DE: Complexes of the minor groove of DNA. Annu Rev Biophys Biomo/ Struct 1995, 24:463-493.
26.
8.
Breslauer KJ, Remata DP, Chou W-Y, Ferrante R, Curry J, Zaunczkowski D, Snyder JG, Marky LA: Enthalpy-entropy compensations in drug-DNA binding studies. Proc Nat/Acad Sci USA 1987, 84:8922-8926.
MrksichM, Dervan PB: Recognition in the minor groove of DNA at 5'-(,AT)GCGC(,AT)-3' by a four ring tripeptide dimer. Reversal of the specificity of the natural product distamycin. J Am Chem Soc 1995, 117:3325-3332.
2?.
9.
Abu-Daya A, Brown PM, Fox KR: DNA sequence preferences of several AT-selective minor groove binding ligands. Nucleic Acids Res 1995, 23:3385-3392.
White S, Baird EE, Dervan PB: Effects of the A.T/T.A degeneracy of th pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 1996, 35:12532-1253Z
28.
10.
Drew HR, Wing RM, Takano T, Broka C, Tanaka S, Itakura K, Dickerson RE: Structure of a B-DNA dodecamer: conformation and dynamics. Proc Nat/Acad Sci USA 1981,78:2179-2183.
Youngquist RS, Dervan PB: Sequence-specific recognition of B-DNA by oligo(N-methylpyrrole-carboxamide)s. Proc Nat/Acad Sci USA 1955, 82:2565-2569.
29.
Youngquist RS, Dervan PB: A synthetic peptide binding 16 base pairs of ,AT double helical DNA. J Am Chem Soc 1987, 109:7564-7566.
11.
NelsonHCM, Finch JT, Luisi BR, Klug A: The structure of an oligo(dA).oligo(dT) tract and its biological implications. Nature 1987, 330:221-226.
30.
12.
Chuprina VB, Lipanov AA, Federoff OYu, Kim SG, Kintanar A, Reid BR: Sequence effects on local DNA topology. Proc Nat/ Acad Sci USA 198'7, 88:9087-9091.
MrksichM, Dervan PB: Design of a covalent peptide heterodimer for sequence-specific recognition in the minor groove of double helical DNA. J Am Chem Soc 1994, 116:3663-3664.
31.
Dwyer TJ, Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Structural analysis of covalent peptide dimers, bis(pyridine2-carboxamidonetropsin)(CH2)3_ 6 in complex with 5'-TGACT3" sites by two dimensional NMR. J Am Chem Soc 1993, 115:9900-9906.
13.
Kopka ML, Yoon C, Goodsell D, Pjura P, Dickerson RE: Binding of an antitumor drug to DNA. J Mo/Bio/1985, 183:553-563.
14.
Coil M, Frederick CA, Wang AH-J, Rich A: A bifurcated hydrogen-bonded conformation in the d(A.T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin. Proc Nat/Acad Sci USA 198"7, 84:8385-8389.
32.
Pelton JG, Wemmer DE: Structural characterization of a 2:1 distamycin A-d(CGCAAATTGGC) complex by two-dimensional NMR. Proc Nat/Acad Sci USA 1989, 86:5723-5?27.
MrksichM, Parks ME, Dervan PB: Hairpin peptide motif. A new class of oligopeptides for sequence-specific recognition in the minor groove of double-helical DNA. J Am Chem Soc 1994, 116:7983-7988.
33.
ParksME, Baird EE, Dervan PB: Optimization of the hairpin polyamide design for recognition of the minor groove of DNA. J A m Chem Soc 1996, 118:6147-6152.
34.
Swalley SE, Baird EE, Dervan PB: Recognition of a 5"-(A,T)GGG(,AT)2-3' sequence in the minor groove of DNA by an eight-ring hairpin polyamide. J Am Chem Soc 1996, 118:8198-8206.
35.
Cho J, Parks ME, Dervan PB: Cyclic polyamides for recognition in the minor groove of DNA. Proc Nat/Acad Sci 1995,
15.
16.
17.
RentzeperisD, Marky LA, Dwyer TJ, Geierstanger BH, Pelton JG, Wemmer DE: Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure. Biochemistry 1995, 34:2937-2945. WemmerDE, Geierstanger BH, Fagan PA, Dwyer TJ, Jacobsen JP, Pelton JG, Ball GE, Leheny AR, Chang WH, Bathini Y e t aL: Minor groove recognition of DNA by distamycin and its analogs. In Structural Biology: The State of the Art, Proceedings of the 8th Conversation. Edited by Sarma RH, Sarma MH. Albany, NY: Adenine Press; 1994:301-323.
18.
Chen X, Ramakrishnan B, Rao ST, Sundaralingam M: Binding of two Distamycin A molecules in the minor groove of an alternating B-DNA duplex. Nat Struct Biol 1994, 1:169-175.
19.
Kopka ML, Yoon C, Goodsell D, Pjura P, Dickerson RE: The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc Nat/Acad Sci USA 1985, 82:1376-1380.
92:10389-10392.
36.
TraugerJW, Baird EE, Mrksich M, Dervan PB: Extension of sequence-specific recognition in the minor groove of DNA by pyrrole-imidazole polyamides to 9-13 base pairs. J Am Chem Soc 1996, 118:6160-6166. This paper describes extended binding motifs that show that sequencespecific recognition can be extended to rather long target sequences. •
37. •
Geierstanger BH, Mrksich M, Dervan PB, Wemmer DE: Extending the recognition site of designed minor groove binding molecules. Nat Struct Bio/1996, 3:321-324.
Targeting the minor groove of DNA Wemmer and Dervan
361
A structural characterization of the 'slipped' extended binding motif is presented, which shows the importance of linkers, and the good fit of ligand and DNA throughout the complex.
42.
Walker S, Murnick J, Kahne D: Structural characterization of a calicheamicin-DNA complex by NMR. J Am Chem Soc 1994, 115:7954-7961.
38. **
43.
Ikemoto N, Kumar RA, Ling l-r, Ellestad GA, Danishefsky SJ, Patel DJ: Calicheamicin-DNA complexes- warhead alignment and saccharide recognition of the minor groove. Proc Nat/Acad Sci USA 1995, 92:10506-10510.
44.
Paloma LG, Smith JA, Chazin WJ, Nicolaou KC: Interaction of calicheamicin with duplex D N A - r o l e of the oligosaccharide domain and identification of multiple binding modes. J Am Chem Soc 1994, 116:369?-3?08.
45.
KahneD: Strategies for the design of minor groove binders: a reevaluation based on the mergence of site-selective carbohydrate binders. Chem Bio/1995, 2:9-12.
46.
Bifulco G, Galeone A, Gomezpaloma L, Nicolaou KC, Chazin WJ: Solution structure of the head-to-head dimer of calicheamicin oligosaccharide domain and d(CGTAGGATATCCTACG)2. J Am Chem Soc 1996, 118:881 ?-8824.
47.
Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB: Regulation of gene expression by small molecules, Nature 1997, in press.
Trauger JW, Baird EE, Dervan PB: Recognition of DNA by designed ligands at subnanomolar concentrations. Nature 1996, 382:559-561. The authors show that four ring hairpin ligands bind with very high affinity and good specificity. 39.
Trauger JW, Baird EE, Dervan PB: Extended hairpin polyamide , motif for sequence-specific recognition in the minor groove of DNA. Chem Bio/1996, 3:369-377. This paper shows that one of the many possible extensions of the extended binding motif is functional, giving high affinity and specificity. 40. o-
Baird EE, Dervan PB: Solid phase synthesis of polyamides containing imidazole and pyrrole amino acids. J Am Chem Soc 1996, 118:6141-6146. This paper describes the synthetic enhancements that make practical the exploration of the binding motifs of polyamides and their applications. 41.
Gao X, Mirau P, Patel DJ: Structure refinement of the chromomycin dimer-DNA oligomer complex in solution. J Mo/ Bio/1992, 223:259-279.