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1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
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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.
Keywords: Bioengineering, Issue 59, molecular machine, bio-nanotechnology, 5'OH DNA breaks, 5'PO4 DNA breaks, apoptosis labeling, in situ detection, vaccinia topoisomerase I, DNA breaks, green nanotechnology
Minchew, C. L., Didenko, V. V. In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage. J. Vis. Exp. (59), e3628, doi:10.3791/3628 (2012).
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.
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
2. Molecular machines assembly
All reagents are scaled for 25 μL total volume, which is sufficient for a single detection in an average size tissue section (10x10mm). The volume can be scaled up as needed.
3. Using molecular machines in tissue sections to dual label 5'OH and 5'PO4 DNA breaks
4. Representative Results
Figure 1. Semi-artificial molecular machine detects two types of DNA damage in situ. The fluorescent machine self-assembles when VACC TOPO binds to the double-hairpin 32-mer. The machine begins operation by splitting itself into two detector units via topoisomerase-made cut at the 3' end of the recognition sequence. This results in a cyclic process where the FITC-labeled unit continuously separates and religates back to the rhodamine-labeled unit. This persists until a detectable DNA break is encountered. When such an alternative acceptor (5'OH blunt end DNA break) is present in the tissue section, the FITC part will ligate to it. The remaining rhodamine part will attach to 5' PO4 DNA blunt end breaks with the help of T4 DNA ligase. Consequently, both types of DNA breaks are simultaneously detected.
Figure 2. Molecular machine dual labels two types of DNA breaks in a tissue section of dexamethasone-treated thymus. Blunt-ended DNA breaks of DNase I- and DNase II-type are detected in the thymic cortical areas undergoing apoptosis. Green cytoplasmic fluorescence (5'OH DNA breaks) marks cortical macrophages digesting nuclear material of apoptotic thymocytes. This signal is produced by VACC TOPO, and localizes to phagolysosomes with DNA containing 5'OH double-strand breaks 11,12. Red fluorescence (5'PO4 breaks) labels nuclei of apoptotic thymocytes not engulfed by macrophages. Massive numbers of thymocytes simultaneously undergo apoptosis accompanied by generation of 5'PO4 double-strand breaks, visualized by ligase-based labeling. These breaks are located at the nuclear periphery, forming ring-shaped patterns. All cellular nuclei are visualized by counterstaining with DAPI (blue fluorescence).
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 vivo nanosensors: 1. the difficulty of making truly uniform and reproducible nano-constructs by using traditional nanomaterials; and 2. the potential toxicity, and high biological reactivity of nanoprobes, particles and other highly-dispersed nanomaterials. The sensor integrates a naturally-occurring molecular machine with artificial components which re-direct it to a new function of DNA damage labeling. The product of such integration is a semi-artificial molecular machine targeted to a new task. While topoisomerases, polymerases and other enzymatic machines are frequently used in biochemical research, they are not integrated with engineered components into individual molecular assemblies. Consequently in their routine use, they are not employed as semi-artificial devices 12.
Here we show how to use the sensor in the tissue section format for simultaneous labeling of DNase I- and DNase II-type breaks. Presently there are no other methods available to perform such simultaneous dual detection.
DNase I- and DNase II-type breaks are important markers of cell death progression, specifically labeling the apoptotic self-autonomous and phagocytic phases 11,16. The sensor is a useful addition to the biomedical research arsenal dealing with the detection and detailed characterization of apoptosis.
No conflicts of interest declared.
This research was supported by grant R01NS062842 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (V.V.D.) and by grants R21 NS064403 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health through ARRA (V.V.D.) and R21 EB006301 National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health (V.V.D.).
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