Affinity maturation of an antibody for the UV-induced DNA lesions 6,4 pyrimidine-pyrimidones

Applied Microbiology and Biotechnology
Bingjie KongHaiying Hang

Abstract

DNA lesions, associated mostly with minor changes in DNA structure, may induce permanent change in heritable coding information. Biochemically, these minor structural changes are difficult to be explored for generating high-affinity antibodies to detect specific DNA lesions in varying sequence contexts. Herein, we established a platform of bacterial display to facilitate antibodies to be matured with high affinity and high specificity against DNA lesions. To achieve this goal, we, for the first time, developed a two-round mutation/screening strategy: (1) using multiple lesion-containing DNA probes for primary maturation and (2) using single lesion-containing DNA probes for second maturation. Specifically, we capitalized on 64M-2 as a parental template to improve affinity for 6-4PP by 710-fold, compared with the model one. In addition, the matured antibody (9c3) is found to be much less dependent on the bases surrounding 6-4PPs than the model one. The mechanistic study from both computational simulation and reverse mutations revealed the critical roles of the two-round mutations in the enhanced binding affinity and independence of surrounding bases. This selection strategy opens a new way to improve affinity and specificity of a...Continue Reading

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Datasets Mentioned

BETA
GM00637

Methods Mentioned

BETA
antibody display
flow
surface plasmon resonance
PCR
flow cytometry
Protein A
chip
confocal microscopy
ELISA
Y52H

Software Mentioned

Agilent MassHunter Workstation
Astra
Sort
Biacore T100
CISRR

Related Concepts

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Antibody Specificity

Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.

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