Stabilizing DNA–Protein Co-Crystals via Intra-Crystal Chemical Ligation of the DNA

Abigail Orun,Ananya Vajapayajula,Christopher Snow,Sara Dmytriw

doi:10.3390/cryst12010049

Abstract

Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond structural biology, biomolecular crystals should be made robust against harsh conditions. To overcome this challenge, we optimized chemical DNA ligation within a co-crystal. Co-crystals from two distinct DNA-binding proteins underwent DNA ligation with the carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) under various optimization conditions: 5′ vs. 3′ terminal phosphate, EDC concentration, EDC incubation time, and repeated EDC dose. This crosslinking and DNA ligation route did not destroy crystal diffraction. In fact, the ligation of DNA across the DNA–DNA junctions was clearly revealed via X-ray diffraction structure determination. Furthermore, crystal macrostructure was fortified. Neither the loss of counterions in pure water, nor incubation in blood serum, nor incubation at low pH (2.0 or 4.5) led to apparent crystal degradation. These findings motivate the use of crosslinked biomolecular co-crystals for purposes beyond structural biology, including biomedical applications.

Highlights

Beyond serving as the fundamental components of life, proteins and DNA are key building blocks for nanoscale self-assemblies
We show that EDC crosslinking dramatically increases crystal stability at the macroscale and does not prevent destroy the crystal nanostructure
To show foundational feasibility for biomedical applications, we demonstrated that crosslinked co-crystals remain robust in aqueous environments, blood serum, and at pH values found in the stomach or lysosomes

Summary

Introduction

Beyond serving as the fundamental components of life, proteins and DNA are key building blocks for nanoscale self-assemblies. Downstream applications of interest, including X-ray diffraction, are hindered by crystal fragility and intolerance to solvent conditions other than the crystal growth solution. We establish a protocol for the chemical ligation of DNA inside of crystals and we demonstrate structural resilience of crosslinked co-crystals which may further their application utility. While coding DNA sticky base overhangs can drive self-assembly, the non-covalent DNA base stacking interactions and Watson–Crick hydrogen bonds that stabilize the junctions are only stable under specific conditions. Introducing covalent bonds across DNA–DNA interfaces has the potential to dramatically improve crystal macro-structure stability and could improve X-ray diffraction

Methods

Results

Conclusion