Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

Abstract

A design principle for sequence-specific DNA modifiers driven by the specific DNA recognition was proposed based on the kinetic parameters for DNA binding and modification reactions.

Tuning the Reactivity of a Substrate for SNAP‐Tag Expands Its Application for Recognition‐Driven DNA‐Protein Conjugation

“…In a parallel experiment, k off was estimated to be 0.49 s −1 for the dissociation of complex by the addition of competitor DNA, which is in the same order of magnitude with the value obtained for the dissociation of original complex (0.64 s −1 ; Figure S5e and Table S1). The estimated K D of 72 nM given by k off /k on is comparable with the previously reported value of 56 nM estimated by the steady‐state FP titration [46] . From the above‐mentioned kinetic parameters ( k app , k on and k off ) and k app =( k on k cov )/( k off + k cov ), the k cov is estimated to be 0.032 s −1 .…”

“…The preparation procedures are briefly outlined below by taking ZF‐SNAP as a typical example. pET30a/ZF‐SNAP was constructed in a polymerase chain reaction (PCR) using the primer pair [45,46] . ZF‐SNAP was expressed in E. coli BL21(DE3) competent cells with the induction by 1 mM isopropyl‐β‐ d ‐1‐thiolgalactopyranoside (IPTG) for 24 h at 25 °C.…”

“…Among the self-ligating protein tags reported, [43] CLIP-tag and its substrate O 2 -benzylcytosine (BC) were assumed to exhibit the reaction kinetics that satisfy the kinetic criterion for recognition-driven reaction. [46] In fact, three MAs sharing the same CLIP-tag but differed in the DBD efficiently distinguished the BC-tethered specific DNA sequence over the nonspecific one. The result also suggested that MA-SNAP would proceed the crosslinking reaction exclusively governed by the sequence specificity of DBD with a BG derivative displaying appropriate reaction kinetics with SNAP-tag (Figure 1c).…”

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier

“…Xylitol dehydrogenase (XDH) ( Watanabe et al, 2005 ), the second enzyme in the D-xylose metabolic pathway that converts xylitol to xylulose by consuming a cofactor NAD + , was assembled on the dynamic DNA scaffold ( Figure 5A ). The modular adaptor ( Nakata et al, 2015 ; Ngo et al, 2016 ; Nguyen et al, 2017 ; Nguyen et al, 2019 ) stably locates an enzyme of interest at the specific position on DNA scaffold with a covalent linkage between the protein and the scaffold. XDH was fused to the C-terminal of modular adaptor (HG) consisting of the basic leucine zipper protein GCN4 ( Ellenberger et al, 1992 ) and Halo-tag ( England et al, 2015 ) to construct a fusion enzyme HG-XDH as reported previously ( Lin et al, 2021 ).…”

“…XDH was fused to the C-terminal of modular adaptor (HG) consisting of the basic leucine zipper protein GCN4 ( Ellenberger et al, 1992 ) and Halo-tag ( England et al, 2015 ) to construct a fusion enzyme HG-XDH as reported previously ( Lin et al, 2021 ). HG-XDH specifically reacts with the Halo-tag substrate 5-chlorohexane (CH) incorporated near the GCN4-binding DNA sequence ( Nguyen et al, 2017 ; Nguyen et al, 2019 ). The dynamic DNA scaffold in the open state was constructed with three hairpin DNAs containing the GCN4-binding DNA sequence modified with CH for HG-XDH ( Supplementary Table S2 ).…”