Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins

David Segal, Roger R. Beerli, Pilar Blancafort, Birgit Dreier, Karin Effertz, Adrian Huber, Beate Koksch, Caren V. Lund, Laurent Magnenat, David Valente, Carlos F. Barbas

Research output: Contribution to journalArticle

127 Citations (Scopus)

Abstract

In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the α-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

Original languageEnglish (US)
Pages (from-to)2137-2148
Number of pages12
JournalBiochemistry
Volume42
Issue number7
DOIs
StatePublished - Feb 25 2003
Externally publishedYes

Fingerprint

Zinc Fingers
DNA-Binding Proteins
Zinc
Proteins
Fingers
Site selection
DNA sequences
DNA
Assays
Technology
Aspartic Acid
Genes
Enzyme-Linked Immunosorbent Assay
Genome
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Segal, D., Beerli, R. R., Blancafort, P., Dreier, B., Effertz, K., Huber, A., ... Barbas, C. F. (2003). Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry, 42(7), 2137-2148. https://doi.org/10.1021/bi026806o

Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. / Segal, David; Beerli, Roger R.; Blancafort, Pilar; Dreier, Birgit; Effertz, Karin; Huber, Adrian; Koksch, Beate; Lund, Caren V.; Magnenat, Laurent; Valente, David; Barbas, Carlos F.

In: Biochemistry, Vol. 42, No. 7, 25.02.2003, p. 2137-2148.

Research output: Contribution to journalArticle

Segal, D, Beerli, RR, Blancafort, P, Dreier, B, Effertz, K, Huber, A, Koksch, B, Lund, CV, Magnenat, L, Valente, D & Barbas, CF 2003, 'Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins', Biochemistry, vol. 42, no. 7, pp. 2137-2148. https://doi.org/10.1021/bi026806o
Segal, David ; Beerli, Roger R. ; Blancafort, Pilar ; Dreier, Birgit ; Effertz, Karin ; Huber, Adrian ; Koksch, Beate ; Lund, Caren V. ; Magnenat, Laurent ; Valente, David ; Barbas, Carlos F. / Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. In: Biochemistry. 2003 ; Vol. 42, No. 7. pp. 2137-2148.
@article{17d6210619bc4e48a6d8792cf7035156,
title = "Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins",
abstract = "In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the α-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.",
author = "David Segal and Beerli, {Roger R.} and Pilar Blancafort and Birgit Dreier and Karin Effertz and Adrian Huber and Beate Koksch and Lund, {Caren V.} and Laurent Magnenat and David Valente and Barbas, {Carlos F.}",
year = "2003",
month = "2",
day = "25",
doi = "10.1021/bi026806o",
language = "English (US)",
volume = "42",
pages = "2137--2148",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins

AU - Segal, David

AU - Beerli, Roger R.

AU - Blancafort, Pilar

AU - Dreier, Birgit

AU - Effertz, Karin

AU - Huber, Adrian

AU - Koksch, Beate

AU - Lund, Caren V.

AU - Magnenat, Laurent

AU - Valente, David

AU - Barbas, Carlos F.

PY - 2003/2/25

Y1 - 2003/2/25

N2 - In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the α-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

AB - In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the α-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

UR - http://www.scopus.com/inward/record.url?scp=0344839044&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0344839044&partnerID=8YFLogxK

U2 - 10.1021/bi026806o

DO - 10.1021/bi026806o

M3 - Article

C2 - 12590603

AN - SCOPUS:0344839044

VL - 42

SP - 2137

EP - 2148

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -