In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Summary

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Abstract

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs ^1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) ^7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments ¹⁰. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one ¹². Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage¹².

Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)^11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO₄ blunt-ended break (DNase I-type breaks)^11,12, if T4 DNA ligase is present in the solution ^13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO₄ blunt-ended breaks, the ligase reacts with 5'PO₄ DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO₄ blunt-ended DNA breaks.

This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

Protocol

The sections for the molecular machine-based detection should be prepared first because their preparation takes more time than the assembly of the molecular device. The construct works well with 5-6μm-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use slide brands which retain sections well, such as ProbeOn Plus charged and precleaned slides (Fisher Scientific) or similar. We recommend at first using a tissue with a well-known pattern of DNA damage which contains both DNase I- and DNase II-type breaks, such as dexamethasone-treated apoptotic rat thymus ^13,14.

1. Preparation of sections

Discussion

In this video, we demonstrate how to assemble and use a dual-labeling DNA damage sensor. The sensor is a molecular machine driven by bio-molecular engine, a virus-encoded protein VACC TOPO linked with artificial components. The presented development exemplifies a bio-enabled approach which advocates adapting biological structures, architectures and actual parts and components of cells to the design of non-toxic molecular scale devices ^12,15. This approach resolves two issues inherent to the field of in viv.......