Quantitative analysis of TALE-DNA interactions suggests polarity effects

Joshua F. Meckler, Mital S. Bhakta, Moon Soo Kim, Robert Ovadia, Chris H. Habrian, Artem Zykovich, Abigail Yu, Sarah H. Lockwood, Robert Morbitzer, Janett Elsäesser, Thomas Lahaye, David Segal, Enoch P. Baldwin

Research output: Contribution to journalArticle

116 Citations (Scopus)

Abstract

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG>HD∼NN NI>NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 50-T. Another surprising observation was that base mismatches at the 50 end of the target site had more disruptive effects on affinity than those at the 30 end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto undescribed polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Original languageEnglish (US)
Pages (from-to)4118-4128
Number of pages11
JournalNucleic Acids Research
Volume41
Issue number7
DOIs
StatePublished - Apr 2013

Fingerprint

DNA
Terminal Repeat Sequences
Electrophoretic Mobility Shift Assay
Transcription Activator-Like Effectors
Reporter Genes
Base Pairing
Transcriptional Activation
Observation
Genome
Amino Acids
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Meckler, J. F., Bhakta, M. S., Kim, M. S., Ovadia, R., Habrian, C. H., Zykovich, A., ... Baldwin, E. P. (2013). Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Research, 41(7), 4118-4128. https://doi.org/10.1093/nar/gkt085

Quantitative analysis of TALE-DNA interactions suggests polarity effects. / Meckler, Joshua F.; Bhakta, Mital S.; Kim, Moon Soo; Ovadia, Robert; Habrian, Chris H.; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H.; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David; Baldwin, Enoch P.

In: Nucleic Acids Research, Vol. 41, No. 7, 04.2013, p. 4118-4128.

Research output: Contribution to journalArticle

Meckler, JF, Bhakta, MS, Kim, MS, Ovadia, R, Habrian, CH, Zykovich, A, Yu, A, Lockwood, SH, Morbitzer, R, Elsäesser, J, Lahaye, T, Segal, D & Baldwin, EP 2013, 'Quantitative analysis of TALE-DNA interactions suggests polarity effects', Nucleic Acids Research, vol. 41, no. 7, pp. 4118-4128. https://doi.org/10.1093/nar/gkt085
Meckler JF, Bhakta MS, Kim MS, Ovadia R, Habrian CH, Zykovich A et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Research. 2013 Apr;41(7):4118-4128. https://doi.org/10.1093/nar/gkt085
Meckler, Joshua F. ; Bhakta, Mital S. ; Kim, Moon Soo ; Ovadia, Robert ; Habrian, Chris H. ; Zykovich, Artem ; Yu, Abigail ; Lockwood, Sarah H. ; Morbitzer, Robert ; Elsäesser, Janett ; Lahaye, Thomas ; Segal, David ; Baldwin, Enoch P. / Quantitative analysis of TALE-DNA interactions suggests polarity effects. In: Nucleic Acids Research. 2013 ; Vol. 41, No. 7. pp. 4118-4128.
@article{a743230c00194cc6be60627f49ea8e53,
title = "Quantitative analysis of TALE-DNA interactions suggests polarity effects",
abstract = "Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG>HD∼NN NI>NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 50-T. Another surprising observation was that base mismatches at the 50 end of the target site had more disruptive effects on affinity than those at the 30 end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto undescribed polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.",
author = "Meckler, {Joshua F.} and Bhakta, {Mital S.} and Kim, {Moon Soo} and Robert Ovadia and Habrian, {Chris H.} and Artem Zykovich and Abigail Yu and Lockwood, {Sarah H.} and Robert Morbitzer and Janett Els{\"a}esser and Thomas Lahaye and David Segal and Baldwin, {Enoch P.}",
year = "2013",
month = "4",
doi = "10.1093/nar/gkt085",
language = "English (US)",
volume = "41",
pages = "4118--4128",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "7",

}

TY - JOUR

T1 - Quantitative analysis of TALE-DNA interactions suggests polarity effects

AU - Meckler, Joshua F.

AU - Bhakta, Mital S.

AU - Kim, Moon Soo

AU - Ovadia, Robert

AU - Habrian, Chris H.

AU - Zykovich, Artem

AU - Yu, Abigail

AU - Lockwood, Sarah H.

AU - Morbitzer, Robert

AU - Elsäesser, Janett

AU - Lahaye, Thomas

AU - Segal, David

AU - Baldwin, Enoch P.

PY - 2013/4

Y1 - 2013/4

N2 - Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG>HD∼NN NI>NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 50-T. Another surprising observation was that base mismatches at the 50 end of the target site had more disruptive effects on affinity than those at the 30 end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto undescribed polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

AB - Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG>HD∼NN NI>NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 50-T. Another surprising observation was that base mismatches at the 50 end of the target site had more disruptive effects on affinity than those at the 30 end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto undescribed polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

UR - http://www.scopus.com/inward/record.url?scp=84876557961&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84876557961&partnerID=8YFLogxK

U2 - 10.1093/nar/gkt085

DO - 10.1093/nar/gkt085

M3 - Article

C2 - 23408851

AN - SCOPUS:84876557961

VL - 41

SP - 4118

EP - 4128

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 7

ER -