В пробирке Ассамблеи Semi-искусственные молекулярные машины и его использование для обнаружения повреждений ДНК

Summary

Мы демонстрируем сборки и применения молекулярного масштаба устройство питается от топоизомеразы белка. Конструкция био-молекулярных датчиков, какие метки два основных типа разрывов ДНК в срезах тканей путем присоединения двух различных флуорофоров в их конец.

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

Разделы для молекулярной машины основе обнаружения должен быть подготовлен, так как их подготовка занимает больше времени, чем сборка молекулярные устройства. Построить хорошо работает с 5-6 мкм толщиной разделы, вырезанные из параформальдегида-фиксированных, парафин блоков ткани. Ис?…

Discussion

В этом видео показано, как собрать и использовать двойной маркировки повреждений ДНК датчика. Датчик молекулярная машина управляется био-молекулярных двигателей, вирус-закодированного белка VACC ТОПО связаны с искусственным компонентов. Представлен пример развития био-включен подход,…

Materials

1. Xylene
2. 80 and 96% Ethanol.
3. 2% solution of bovine serum albumin (BSA) in distilled water.
4. Oligonucleotide 1: a double-hairpin, dual labeled with fluorescein and tetramethylrhodamine:

5′-AAG GGA CCT GCF GCA GGT CCC TTA ACG CAT RAT GCG TT- 3′; F – FITC-dT; R – Tetramethylrhodamine-dT. Other red-shifted fluorophores such as BODIPY TR, rhodamine or TAMRA can be used instead of tetramethylrhodamine as R.

Alternatively you can use Oligonucleotide 2 – a double-hairpin single labeled with fluorescein (for detection of a single type of DNA breaks (DNase II-type only). The single-fluorophore-carrying probe is considerably less expensive and is convenient whenever a single type of DNA break is to be labeled: 5′-AAG GGA CCT GCF GCA GGT CCC TTA ACG CAT ATG CGT T-3′; F – FITC-dT

5. Oligonucleotide 3. Blunt-ended rhodamine-labeled hairpin for enhancement of in situ ligation signal with T4 DNA ligase: 5′-GCG CTA GAC CRG GTC TAG CGC-3′; R – Tetramethylrhodamine-dT
6. Vaccinia DNA topoisomerase l (VACC TOPO) – 3000 U/μL (Vivid Technologies). In the initial experiments we used 215 pmoles (7.1 μg) of the VACC TOPO per every 25 μL of the reaction mix. However, the topoisomerase concentration can be significantly reduced without the loss of sensitivity. We later used a four times lesser amount of VACC TOPO per section (1.76 μg in 25 μL of the reaction mix per section) with similar results. Reducing amount of the enzyme to 880 ng (in 25 μL of the reaction mix) resulted in a weaker signal and 8.8 ng of VACC TOPO produced no detectable signal.
7. T4 DNA ligase 5 U/μL (Roche). This highly concentrated ligase preparation gives the best signal in our experimental conditions.
8. 10 x reaction buffer for T4 DNA ligase: 660 mM Tris-HCl, 50 mM MgCl₂, 10 mM dithioerythritol, 10 mM ATP, pH 7.5 (20° C) (Roche) . ATP in reaction buffer is easily destroyed in repetitive cycles of thawing-freezing. Aliquot the buffer in small 15-20 μL portions and store at – 20° C. Use once.
9. 30% (w/v) solution of PEG-8000 (Sigma) in bidistilled water. 15 % PEG-8000 in the reaction mix strongly stimulates the detection, increasing the effective concentrations of its constituents by volume exclusion.
10. Proteinase K (Roche) 20 mg/mL stock solution in distilled water. Store at – 20° C. In the reaction use 50mg/mL solution in PBS, prepared from the stock. Do not reuse.
11. Vectashield with DAPI (Vector Laboratories).
12. Sodium bicarbonate buffer: 50mM NaHCO₃, 15mM NaCl, pH 8.2.
13. 22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.