Abstract
Transcription activator-like effectors (TALEs) are DNA major-groove binding proteins widely used for genome targeting. TALEs contain an N-terminal region (NTR) and a central repeat domain (CRD). Repeats of the CRD selectively recognize each one DNA nucleobase, offering programmability. Moreover, repeats with selectivity for 5-methylcytosine (5mC) and its oxidized derivatives can be designed for analytical applications. However, both TALE domains also nonspecifically interact with DNA phosphates via basic amino acids. To enhance the 5mC selectivity of TALEs, we aimed to decrease the nonselective binding energy of TALEs. We substituted basic amino acids with alanine in the NTR and identified TALE mutants with increased selectivity. We then analysed conserved, DNA phosphate-binding KQ diresidues in CRD repeats and identified further improved mutants. Combination of mutations in the NTR and CRD was highly synergetic and resulted in TALE scaffolds with up to 4.3-fold increased selectivity in genomic 5mC analysis via affinity enrichment. Moreover, transcriptional activation in HEK293T cells by a TALE-VP64 construct based on this scaffold design exhibited a 3.5-fold increased 5mC selectivity. This provides perspectives for improved 5mC analysis and for the 5mC-conditional control of TALE-based editing constructs in vivo.
Highlights
We have recently reported the use of engineered transcription activator-like effectors (TALEs24,25) as programmable DNA binding domains for the analysis of 5mC and other epigenetic cytosine 5-modifications[26,27]
Though the full-length CTR of several Transcription activator-like effectors (TALEs) scaffolds contains multiple of such residues and a high positive net charge, we previously employed TALE scaffolds for 5mC detection that contained a truncated 23 amino acid CTR with only a single basic residue and no positive net charge[39]
The involvement of these residues in DNA binding is further suggested by the findings that a truncated 140 amino acid N-terminal region (NTR) domain without central repeat domain (CRD) and CTR is capable of binding to dsDNA in a sequence-independent manner[40] and that TALEs require at least a minimal NTR fragment to reach full activity in the form of different fusion constructs[45,46,47,48]
Summary
We have recently reported the use of engineered transcription activator-like effectors (TALEs24,25) as programmable DNA binding domains for the analysis of 5mC and other epigenetic cytosine 5-modifications[26,27]. TALEs consist of an N-terminal region (NTR), a C-terminal region (CTR), and a central domain of regular repeats (CRD) that each selectively recognizes one nucleobase via one of two variable amino acids (repeat variable diresidue, RVD). This recognition occurs via a predictable code with the RVDs NI, NN, NG and HD (amino acid positions 12 and 13 within each TALE repeat) preferentially binding A, G(A), T, and C, respectively[28,29]
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