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Design of novel sequence-specific DNA-binding David J Segal and Carlos F Barbas III*

The design and selection of DNA-binding proteins or individual Helical domains domains capable of novel sequence recognition continues to For proteins that do not contact DNA with an organized make great strides. Recent studies have also highlighted the α-helix, success in generating novel sequence-specific pro- role of the non-DNA-contacting portions of the and the teins has been extremely limited. For example, the optimal assembly of the domains. For the first time, it appears base-specific hydrogen bonds between the restriction that it is possible to produce proteins capable of targeting any endonuclease EcoRV and its recognition base pairs are with an 18 base pair recognition domain. A variety of made through a highly cooperative set of interactions by applications are being explored, such as targeted five residues in a surface loop of the enzyme (Figure 1a; transcriptional regulation, recombination and viral integration. [1]). Through application of DNA shuffling, mutagenesis These proteins will probably find diverse applications in gene and screening, Pingoud and co-workers [2••] were able to therapy, functional genomics, and agriculture. extend recognition to include the base pairs immediately flanking the EcoRV site. Although this is an impressive Addresses study, it remains to be seen if more can be expected from The Skaggs Institute for Chemical Biology and the Department of these types of complex recognition motifs. Molecular Biology, The Scripps Research Institute, BCC-515, North Torrey Pines Road, La Jolla, CA 92037, USA A decade ago, the dimeric bZIP proteins were proposed as *e-mail: [email protected] candidates for the development of novel DNA-binding Current Opinion in Chemical Biology 2000, 4:34–39 proteins [3]. Each bZIP monomer contacts DNA with an α 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. -helix that is oriented and constrained by being part of All rights reserved. longer α-helix, which makes up the dimer- ization interface (Figure 1b; [4]). In vitro selection methods Abbreviations DBD DNA- produced mutants of the bZIP protein C/EBP that could ER estrogen recognize sequences that differed in two of its five half-site PR nucleotides [5]. Interestingly, some of the mutations occurred in the hinge region between the α-helix of the Introduction DNA-binding domain (DBD) and the leucine zipper The design of proteins with the capacity to recognize framework, suggesting that reorientation of the α-helix extended nucleotide sequences with high affinity and may have been required. specificity has been a goal of two decades of research; a goal that has remained elusive until the past two years. Theoretically, DBDs of bZIP heterodimers, such as Jun- Ideally, proteins could be designed to specifically regu- Fos, could be modified to recognize novel 10-nucleotide, late the of a single gene in a genome as non-palindromic sites. Alternatively, tandem monomers of complex as our own four billion base pair genome. These the bZIP DBDs could potentially be used to recognize proteins would allow scientists to reach into genomes extended sequences, but these typically bind poorly to and to modulate the transcription of a gene of interest, to DNA. In an elegant design approach, the affinity of a bZIP assign its function or to change the phenotype of monomer DBD, that of , was improved 270-fold by the organism. grafting the DNA-contacting residues from the α-helix of GCN4 onto the stable and compact scaffold of the avian Specific recognition of double-stranded DNA by pro- pancreatic polypeptide [6••]. This miniature 42-amino- teins is most typically accomplished using an α-helix as acid protein, consisting of a single amphipathic α-helix the key recognition element. An α-helix provides a rigid that is stabilized through hydrophobic interactions with a scaffold from which amino-acid sidechains are presented type II polyproline helix, was able to bind a GCN4 half site to the accessible edges of the bases in the major grove of with an affinity and specificity greater than that of the DNA. This presentation is modulated by ‘framework’ parental α-helix. This study again illustrates that binding structural elements of the protein. For proteins in which can be improved by optimizing not only the DNA-contact- the α-helix is the key recognition element, the task of ing residues but also the display and stability of the creating DNA-binding proteins with novel sequence α-helix. It remains unclear if these domains can be effec- specificity consists of modifying the DNA-contacting tively linked as tandem multimers to recognize extended residues in the α-helix, then modifying the framework to sites. Chemical linkage of two mini-proteins provided a optimize the display of the α-helix. In this review, we ‘dimer’ with increased affinity but lower specificity [6••]. discuss the recent successful application of these princi- ples to create novel DNA-binding proteins, with Nuclear receptors, such as the (ER) particular emphasis on the advances achieved using and progesterone receptor (PR), also bind as dimers to α Cys2-His2 finger proteins. their DNA targets. Each monomer inserts an -helix of a ch4106.qxd 02/15/2000 09:01 Page 35

Design of novel sequence-specific DNA-binding proteins Segal and Barbas 35

Figure 1

DBDs that have been used to create proteins with novel sequence specificity. (a) EcoRV (a) (b) [1]. (b) bZIP of GCN4 [4]. (c) Cys4 zinc fingers of [8]. (d) Helix-turn-helix domains of Myb [10]. (e) Cys2–His2 zinc fingers of Zif268 [13]. Double stranded DNA is shown in orange and brown, DBD domains are shown in red, blue or green ribbon representation, and zinc ions are shown as yellow spheres. (d)

(c) (e)

Current Opinion in Chemical Biology

Cys4-type into the major grove, and for some By any measure, the greatest success in producing proteins receptors the DBD contains a strong dimerization inter- with novel binding specificity has been achieved with the face (Figure 1c; [7,8]). Using in vivo survival-based classic Cys2–His2 zinc-finger domains (Figure 1e). These selection of randomized libraries, Shapiro and co-workers zinc fingers are compact domains containing a single [9••] found PR-DBD mutants that lost their ability to rec- amphipathic α-helix stabilized by two β-strands and zinc ognize the PR response element (PRE) but could bind ligation [13]. Like the Myb domains, zinc-finger proteins the ER response element (ERE), which differs in two of contain multiple tandem repeats and display varying the six half-site nucleotides. The mutants bound the degrees of inter-domain cooperativity. Fortunately, a subset ERE with a 15-fold higher affinity than wild-type ER- of zinc-finger proteins, including the murine transcription DBD, albeit with a specificity considerably broader than factor Zif268 and the human protein Sp1, display only min- wild-type ER-DBD. imal — though still troublesome — cooperativity. In these proteins, each finger recognizes a three nucleotide site with From domains to proteins relative independence, which has allowed several groups to Tandemly repeated elements, although having the potential produce zinc fingers with novel specificities using rational for extended, non-palindromic recognition, introduce anoth- or combinatorial methods (reviewed in [14••]). er level of complexity: inter-domain cooperativity. One domain may make protein–protein contacts with the next Early attempts to produce zinc fingers with novel domain, affect the binding geometry of adjacent domains, or sequence recognition gave hope to the idea that there it may contact the nucleotides of another domain’s binding might be a simple 1:1 amino acid to base recognition code site. These interactions that allow for concerted recognition that could be used to build fingers that could specifically may be considered as evolutionary levers for optimizing the recognize any three nucleotide sequence [15,16]. binding of a particular protein; however, for those seeking to Unfortunately, this simplistic code is poorly predictive of design novel binding proteins these interactions present the actual specificity of these domains [17,18•]. In one additional challenges. For example, Myb domains report of a code-based three-finger protein, only five of the (Figure 1d) consist of two or three tandem repeats (desig- nine nucleotides appear to be correctly specified [17]. nated R1, R2 and R3) of a helix-turn-helix motif, similar to the motif found in the λ repressor and homeodomian pro- There are at least three reasons why a simple code is insuf- teins [10]. Myb DBDs with novel specificity have been ficient for comprehensive recognition. The first is generated by combining the R2 and R3 of different species technical. has proven to be a powerful tool [11]; however, the combination of two tandem R3 repeats for revealing the repertoire of zinc-finger–DNA interac- severely reduced binding affinity [12]. The authors con- tions. This procedure involves the display of a large cluded that cooperative interactions between the R2 and R3 (typically 107–109 member) library of randomized proteins domains were required for high affinity binding. on the surface of a filamentous bacteriophage. Because ch4106.qxd 02/15/2000 09:01 Page 36

36 Interaction, assembly and processing

Table 1 two α-helices including position 2 [20•,21], or by replace- ment of the offending domain with a finger that does not Affinities of sequentially-selected and stitched three-finger contain aspartate in position 2 and is not expected to have proteins. any cooperative effects. Protein Target sequence Published Normalized ‡ § KD (nM) KD (nM) Concerns regarding cooperativity can also be addressed by the selection methodology if sequential selections are Stitched study Zif268* GCG TGG GCG 10 10 applied [22]. In this approach (Figure 2a) finger 1 of a B3* GGA GGG GAC 4 4 three-finger protein is combinatorially selected in the con- C5* GGA GGC GGG 30 30 text of two ‘anchor’ fingers. Subsequently, the terminal E2C-HS2(SP1)* GCC GCA GTG 25 25 anchor finger is removed, finger 1 becomes finger 2, and a Sequential study new finger 1 is selected to bind the next three nucleotides Zif268† GCG GGG GCG 0.010 10 in the DNA sequence. After one additional round of † TATA ZF GCT ATA AAA 0.12 120 † # exchanges, a new three-finger protein is created in which p53ZF GGG ACA TGT 0.11 110 † all of the fingers have been selected in the ‘context’ of the NREZF AAG GGT TCA 0.038 38 finger next to it. This is in contrast to direct selection of *Data from [23••]. †Data from [22]. ‡Standard deviation < 60%. § # defined fingers from a single library that are then ‘stitched’ Published values normalized to Zif268 = 10 nM. p53ZF was later • reported to bind with an affinity higher than NREZF [18 ]. together (or at least the contact residues) to make a new three-finger protein (Figure 2b). The advantage of the stitchery method is that once fingers of high affinity and each phage contains the gene for the protein it displays, specificity are created, they can be rapidly reassembled to the sequence of proteins having the desired properties can recognize any desired sequence. However, this aim be identified through repeated cycles of in vitro selection requires that each finger be completely modular and inde- and amplification of the phage library (known as ‘pan- pendent. On the other hand, sequential selection requires ning’). There is, however, a common misconception that that three sequential libraries be constructed and selected any sequence that is strongly selected from panning must for each three-finger protein, making zinc-finger technolo- be the optimal sequence. The flaw in that logic is that the gy inaccessible to most researchers. researcher is not always aware of what the actual selection pressure is during an experiment. Our approach [14••] has Is sequential selection necessary? Several stitched three- been to optimize the output of phage display using site- finger proteins have been constructed from an optimized directed mutagenesis. This study revealed that some set of GNN-binding fingers [14••,23••]. In these fingers, residues were selected during panning because they cooperativity effects caused by aspartate in position 2 are increased the affinity of the interaction at the cost of speci- minimized as all of the target sites are of the GNN type. ficity. The end product of this study was a collection of 16 The published specificity of sequentially selected proteins well-characterized domains recognizing each of the possi- appears to be no better than that of the stitched proteins ble GNN binding sites. A number of these domains [18•,23••]. The affinity of the sequentially selected pro- demonstrated exquisite specificity and discriminated teins was reported to be in the sub-nanomolar range [22]. between sequences that differ by one in nine bases with Regrettably, however, the issue of affinity has become > 100-fold loss in affinity. somewhat distorted in the zinc-finger field. As one exam- ple, the affinity of Zif268 for its operator ‘improved’ Sequential selection versus stitchery 600-fold by modification of the binding measurements The second reason for the problems with simple coded inter- [13,22]. Although the use of non-standard binding condi- actions relates to cooperativity. A central theme in recent tions when measuring affinities is legitimate, the values zinc-finger research has been to refocus on the question: just obtained can not be directly compared to affinities mea- how independent are the zinc fingers of Zif268? An early sured in a more conventional way. A more accurate clue was the difficulty that every laboratory encountered in comparison can be obtained by normalizing the published selecting fingers that could recognize sequences of the type affinities to a common value for Zif268 as this measure- ANN or CNN, instead of GNN — the sequence type recog- ment is also reported. As shown in Table 1, the nized by the natural protein. With Zif268 as their scaffold sequentially selected proteins bind their target with a protein, a consensus in the field emerged that aspartate in lower affinity than the stitched proteins. On the basis of position 2 of one α-helix contacted the binding site of the this comparison the advantages of sequential selection are finger next to it, forcing recognition at that neighboring site not clear. The comparison is not entirely fair, however, as to be GNN or TNN [19,20•]. The significance of this con- the sequentially selected proteins contain fingers that rec- tact was underappreciated in the original Zif268 crystal ognize ANN and TNN sites, which may be inherently structure [13] but is now widely acknowledged and consti- more difficult for zinc fingers to bind with high affinity and tutes what is known as the target site overlap problem in zinc specificity. Studies in live cells suggest that specificity is a fingers [14••]. Limitations imposed by this contact can be problem as the TATA protein was not effective in reporter addressed by randomization of residues at the interface of gene studies [24]. ch4106.qxd 02/15/2000 09:01 Page 37

Design of novel sequence-specific DNA-binding proteins Segal and Barbas 37

Figure 2

Sequential and parallel selection methods. Boxes represent zinc finger domains. Letters (a)Sequential selection (b) Parallel selection and stitchery beneath the boxes represent DNA sequences. See text for details. Anchor Anchor Library Anchor Library Anchor GCG TGG ABC DEF GHI GCG ABC GCG

Anchor ABC Library Anchor Library Anchor

GCG TGG ABC DEF GHI GCG DEF GCG

ABC DEF Library Anchor Library Anchor GCG TGG ABC DEF GHI GCG GHI GCG

ABC DEF GHI ABC DEF GHI

Current Opinion in Chemical Biology

Framework effects Additional studies that correlate structure with quantita- A third reason for the problems with simple coded interac- tive affinity and specificity data will be required to further tions is that a code fails to consider the influence of the reveal how framework effects influence the ability of zinc- linker region, the β-strands, and the carboxy-terminal end finger proteins to both recognize specific sequences and of the α-helix (collectively referred to as the framework). exclude highly related ones. Framework effects on specificity and affinity are only beginning to be appreciated and will undoubtedly repre- Despite these complexities, a polydactyl protein specifi- sent a new area of research in this field. Structures of cally recognizing an 18-nucleotide sequence in the Zif268 mutants binding their designed targets clearly show 5′-untranslated region of the human erbB-2 gene has been a repositioning of the α-helix relative to the DNA, sug- constructed. This protein bound the target sequence with gesting that the α-helix might require reorientation for the 0.5 nM affinity. When expressed as a fusion protein with optimal presentation of its contacting residues to variant repressor or domains, the was DNA sites [25••]. Variation in helical presentation is also able to specifically regulate the erbB-2 promoter [23••]. clearly evident in recent structural studies of three-finger Extension of this work to the study of the regulation of the and six-finger proteins derived from TFIIIA [26,27]. In our endogenous erbB-2 gene has resulted in the first demon- own studies [23••], a 50-fold increase in affinity was stration of the modulation of endogenous with observed when the same six α-helices were displayed on designed transcription factors (CF Barbas III, unpublished different frameworks. Ryan and Darby [28] reported an data; see Note added in proof). These proteins were shown eightfold loss in affinity and a 50-fold loss in specificity to be effective in cells derived from three mammalian after modifying only the linker regions of TFIIIA. species that conserve the erbB-2 binding site. Amazingly, the erbB-3 gene is not regulated despite a 15 of 18 Appropriate display of the α-helix becomes particularly nucleotide match to the erbB-2 target site at the same - important as the number of tandem domains is increased tive position. Furthermore, construction of an to six, the number required to recognize a unique erbB-3-targetting transcription factor enables specific reg- sequence in the human genome [29]. No natural zinc fin- ulation of the gene as well. Thus it appears that ger proteins have been found that bind specifically to 18 genome-specific regulation is now in reach. contiguous nucleotides. Additionally, the DNA duplex is unwound slightly upon binding, causing concern that the Conclusions contact residues in a long protein may come out of register With the demonstration that polydactyl zinc-finger pro- with the nucleotides, resulting in a loss in specificity and teins can be readily assembled to recognize 18 base-pair •• ′ ′ affinity. Our studies [23 ,29] suggest that the affinity of a sequences of the 5 -(GNN)6-3 type, and that these pro- six-finger protein is highly dependant on the framework. teins can specifically regulate an endogenous gene, much In another example, the use of an 11-amino-acid linker additional study will be needed to extend the type of peptide produced a six-finger protein with an affinity sequences that can be addressed by this approach. Some > 6000-fold greater than that of its three-finger compo- sequence motifs may require the use of other DNA-bind- nents [30•]. Framework mutations have tended to be ing proteins or combinations of domains of different types. context-dependent in the zinc finger area and application This might be accomplished through the selection of novel of this 11-amino-acid linker to other proteins has not dimerizing regions that serve to link varied proteins non- resulted in other examples with exceptional affinity. covalently [31••]. Now that we have begun to be able place ch4106.qxd 02/15/2000 09:01 Page 38

38 Interaction, assembly and processing

proteins at specific addresses within complex genomes, tion methods are described. The authors are poised to discover if these Cys4 what activities might these proteins direct? Given the zinc fingers are capable of the versatility of their Cys2–His2 cousins. modular nature of proteins that act on DNA, a vast array of 10. Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H, Sarai A, Ishii S, Nishimura Y: Solution structure of a specific DNA tools that facilitate the manipulation of genomes is possi- complex of the Myb DNA-binding domain with cooperative ble. Appending activation or repression domains onto a recognition helices. Cell 1994, 79:639-648. DBD allows for transcriptional modulation [23••,29], while 11. Williams CE, Grotewold E: Differences between plant and animal • Myb domains are fundamental for DNA binding activity, and endonuclease [32,33 ], methylase [34], topoisomerase [35], chimeric Myb domains have novel DNA binding specificities. and HIV-1 integrase [36] domains have allowed for some J Biol Chem 1997, 272:563-571. degree of site-directed action on DNA. Although many of 12. Oda M, Furukawa K, Sarai A, Nakamura H: Construction of an these novel functions are still too inefficient for practical artificial tandem protein of the c-Myb DNA-binding domain and analysis of its DNA binding specificity. Biochem Biophys Res application, they are certain to have an impact on basic and Commun 1999, 262:94-97. applied biology in this new era of the genome. 13. Pavletich NP, Pabo CO: Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 Å. Science 1991, 252:809-817. Note added in proof 14. Segal DJ, Dreier B, Beerli RR, Barbas CF III: Toward controlling The paper referred to in the text as (CF Barbas III, unpub- •• gene expression at will: selection and design of zinc finger lished data) has now been accepted for publication [37]. domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc Natl Acad Sci USA 1999, 96:2758-2763. The domains reported in this paper were optimized using a combination of References and recommended reading phage display and rational mutagenesis. Domains are disclosed that recog- Papers of particular interest, published within the annual period of review, nize any GNN target sequence (guanine followed by any two bases. Many have been highlighted as: of these domains exhibit exquisite specificities not previously observed for zinc-finger proteins. Together with [23••], this paper describes a system by • of special interest which any laboratory can build a zinc finger protein capable, perhaps, of •• of outstanding interest uniquely targeting any gene in a complex genome. 1. Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, 15. Choo Y, Klug A: Selection of DNA binding sites for zinc fingers Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS: The using rationally randomized DNA reveals coded interactions. Proc crystal structure of EcoRV endonuclease and of its complexes Natl Acad Sci USA 1994, 91:11168-11172. with cognate and non-cognate DNA fragments. EMBO J 1993, 12:1781-1795. 16. Desjarlais JR, Berg JM: Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding 2. Lanio T, Jeltsch A, Pingoud A: Towards the design of rare cutting proteins. Proc Natl Acad Sci USA 1993, 90:2256-2260. •• restriction endonucleases: using directed to generate variants of EcoRV differing in their substrate specificity by two 17. Corbi N, Libri V, Fanciulli M, Passananti C: Binding properties of the orders of magnitude. J Mol Biol 1998, 283:59-69. artificial zinc fingers coding gene Sint1. Biochem Biophys Res This paper describes one of the very few modifications of DNA recognition Commun 1998, 253:686-692. α in an enzyme using a non- -helical recognition motif. DNA shuffling was 18. Wolfe SA, Greisman HA, Ramm EI, Pabo CO: Analysis of zinc used to evolve the specificity of the enzyme to a longer sequence than its • fingers optimized via phage display: evaluating the utility of a native target. Extension of the sequence specificity of restriction enzymes to recognition code. J Mol Biol 1999, 285:1917-1934. longer sequences promises many practical applications in cloning and This paper reports the specificity of the proteins constructed by sequential recombination. selection (see [22]). The authors also argue that many of the interactions 3. O’Neil KT, Hoess RH, DeGrado WF: Design of DNA-binding they observed were not predicted by a recognition code. peptides based on the leucine zipper motif. Science 1990, 19. Isalan M, Choo Y, Klug A: Synergy between adjacent zinc fingers in 249:774-778. sequence-specific DNA recognition. Proc Natl Acad Sci USA 1997, 4. Ellenberger TE, Brandl CJ, Struhl K, Harrison SC: The GCN4 basic 94:5617-5621. region leucine zipper binds DNA as a dimer of uninterrupted 20. Isalan M, Klug A, Choo Y: Comprehensive DNA recognition through α-helices: crystal structure of the protein–DNA complex. Cell • concerted interactions from adjacent zinc fingers. Biochemistry 1992, 71:1223-1237. 1998, 37:12026-12033. 5. Sera T, Schultz PG: In vivo selection of basic region-leucine zipper By randomizing residues at the interface of two domains, the authors proteins with altered DNA-binding specificities. Proc Natl Acad Sci showed that they could select residues that would allow the recognition of USA 1996, 93:2920-2925. sequences starting with any base. It is still not clear if these results can be generalized, however, and the authors have yet to show that they can apply 6. Zondlo NJ, Schepartz A: Highly specific DNA recognition by a their methods to make multi-finger proteins of high affinity and specificity or •• designed miniature protein. J Am Chem Soc 1999, 121:6938- that purified rather than phage-bound proteins actually bind the target 6939. sequence. This study demonstrates minimalist design at its best. Simple grafting of the recognition residues of GCN4 onto the well-studied avian pancreatic 21. Jamieson AC, Wang H, Kim S-H: A zinc finger directory for high- polypeptide and a little fine-tuning provided a small 42-residue protein with affinity DNA recognition. Proc Natl Acad Sci USA 1996, 93:12834- exquisite affinity and specificity for the target DNA, much greater than the 12839. native protein. 22. Greisman HA, Pabo CO: A general strategy for selecting high- 7. Schwabe JW, Chapman L, Finch JT, Rhodes D: The crystal structure affinity zinc finger proteins for diverse DNA target sites. Science of the estrogen receptor DNA-binding domain bound to DNA: how 1997, 275:657-661. receptors discriminate between their response elements. Cell Toward controlling 1993, 75:567-578. 23. Beerli RR, Segal DJ, Dreier B, Barbas CF III: •• gene expression at will: specific regulation of the erbB-2/HER-2 8. Luisi BF, Xu WX, Otwinski Z, Freedman LP, Yamamoto KR, Sigler PB: promoter by using polydactyl zinc finger proteins constructed Crystallographic analysis of the interaction of the glucocorticoid from modular building blocks. Proc Natl Acad Sci USA 1998, receptor with DNA. Nature 1991, 352:497-505. 95:14628-14633. This paper presents the assembly, affinity, specificity, and biological activity 9. Chusacultanachai S, Glenn KA, Rodriguez AO, Read EK, Gardner JF, of six-domain polydactyl zinc-finger proteins using the domains described in •• Katzenellenbogen BS, Shapiro DJ: Analysis of estrogen response [14••]. Transcription factors recognizing an 18 base pair sequence are element binding by genetically selected steroid receptor DNA assembled and demonstrated to specifically control the erbB-2 promoter in binding domain mutants exhibiting altered specificity and human cells, the first time that transcription factors have been fashioned to enhanced affinity. J Biol Chem 1999, 274:23591-23598. regulate a specific promoter. Together, these papers provide the necessary This study is the most extensive analysis of the elements of recognition for tools by which any laboratory can build a zinc finger protein capable, per- Cys4-type zinc-finger binding motifs. Novel randomization and in vivo selec- haps, of uniquely targeting any gene in the human genome. ch4106.qxd 02/15/2000 09:01 Page 39

Design of novel sequence-specific DNA-binding proteins Segal and Barbas 39

24. Kim JS, Pabo CO: Transcriptional repression by zinc finger the effect has anything to do with the noncanonical linker, as it appears that peptides. Exploring the potential for applications in gene therapy. the canonical linker was never tested. Kinetic measurements are used to J Biol Chem 1997, 272:29795-29800. derive dissociation constants. As performed, these measurements can lead to very large errors. Therefore, the use of the term ‘femtomolar’ in the title and 25. Elrod-Erickson M, Benson TE, Pabo CO: High-resolution structures abstract of this paper is provocatively misleading. Normalizing the reported •• of variant Zif268–DNA complexes: implications for understanding affinities to a more traditional value for Zif268 produces dissociation con- zinc finger–DNA recognition. Structure 1998, 6:451-464. stants in the picomolar range. Such affinities are still impressive, but are This paper presents the structures of phage display selected mutants. The achievable with canonical linkers. structures show interactions in cognate and non-cognate sites. The paper also describes the repositioning of the α-helix relative to the DNA. This is an 31. Zhang Z, Murphy A, Hu JC, Kodadek T: Genetic selection of short excellent and necessary descriptive work, although the paucity of quantita- •• peptides that support protein oligomerization in vivo. Curr Biol tive affinity and specificity data for these proteins allows for few conclusions 1999, 9:417-420. regarding specific interactions that provide for binding one sequence while In this study a genetic selection is devised to select novel peptides that excluding others. result in dimerization or multimerization of the λ repressor. Effective peptide motifs allow cell survival following assault with the lytic phage. An amazing- 26. Nolte RT, Conlin RM, Harrison SC, Brown RS: Differing roles for ly diverse selection of novel functional peptides results from this. zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex. Proc Natl Acad Sci USA 1998, 32. Nahon E, Raveh D: Targeting a truncated HO-endonuclease of 95:2938-2943. yeast to novel DNA sites with foreign zinc fingers. Nucleic Acids Res 1998, 26:1233-1239. 27. Wuttke DS, Foster MP, Case DA, Gottesfeld JM, Wright PE: Solution structure of the first three zinc fingers of TFIIIA bound to the 33. Chandrasegaran S, Smith J: Chimeric restriction enzymes: what is • cognate DNA sequence: determinants of affinity and sequence next? Biol Chem 1999, 380:841-848. specificity. J Mol Biol 1997, 273:183-206. This review describes fusions of zinc finger proteins and the gal4 DBD with the endonuclease domain of FokI. The zinc finger proteins were constructed 28. Ryan RF, Darby MK: The role of zinc finger linkers in p43 and entirely by rational design. The fusions exhibit activity, though their ability to TFIIIA binding to 5S rRNA and DNA. Nucleic Acids Res 1998, catalyze multiple DNA cleavages is not assessed. Efficient site-specific 26:703-709. enzymes may be only an evolutionary selection away. 29. Liu Q, Segal DJ, Ghiara JB, Barbas CF III: Design of polydactyl zinc- 34. Xu G-L, Bestor TH: Cytosine methylation targetted to pre- finger proteins for unique addressing within complex genomes. determined sequences. Nat Genet 1997, 17:376-378. Proc Natl Acad Sci USA 1997, 94:5525-5530. 35. Beretta GL, Binaschi M, Zagni E, Capuani L, Capranico G: Tethering 30. Kim JS, Pabo CO: Getting a handhold on DNA: design of poly-zinc a type IB topoisomerase to a DNA site by enzyme fusion to a • finger proteins with femtomolar dissociation constants. Proc Natl heterologous site-selective DNA-binding . Cancer Acad Sci USA 1998, 95:2812-2817. Res 1999, 59:3689-3697. This work suggests that modifications to the framework of a zinc-finger pro- 36. Bushman FD, Miller MD: Tethering human immunodeficiency virus tein can lead to dramatic improvements in binding. The authors state that by > type 1 preintegration complexes to target DNA promotes using a noncanonical linker between zinc fingers 3 and 4, a 6000-fold integration at nearby sites. J Virol 1997, 71:458-464. increase in affinity can be obtained by a six-finger protein over that of its con- stituent three-fingers proteins. It remains to be seen if specificity is sacri- 37. Beerli RB, Dreier B, Barbas CF III: Selective positive and negative ficed, or if this effect can be reproduced with other proteins. To date, further regulation of endogenous genes by designed transcription application of this approach has failed. More importantly, it is not clear that factors. Proc Natl Acad Sci USA 2000, in press.