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

The overall goal of this procedure is to demonstrate how to assemble and use a dual labeling DNA damage sensor that labels two major types of DNA breaks in tissue sections. The sensor is comprised of a 30 tumor, DNA oligonucleotide dual labeled with ZI and Rumine, and an enzyme vaccinia topo isomerase one or vac topo that self assembles. When VAC topo binds to the 30 tumor, the machine operation starts when topo is sommes cleaves the oligonucleotide at the three prime end of the recognition sequence resulting in a vac topo activated fitzy labeled unit and amine labeled free hairpin.

With a terminal three-prime hydroxyl, the units are short-lived and quickly reassembled back into the original construct, which is subsequently cleaved. In the absence of additional extra molecular DNA breaks these two units continuously separate and relegate in a cyclic manner. When the molecular sensor is applied to tissue sections with DNA damage, the Fitz labeled detector unit selectively attaches to blunt ended DNA breaks with five-prime hydroxyl.

The R domine labeled detector unit will ligate to a five-prime phosphate blunt ended break. If T four DNA Ligase is present, results are obtained that show the presence and distribution of DNA's one and D'S two type DNA breaks in cells based on fluorescence microscopy. The main advantage of this technique over existing methods for HESIs detection like tunnel is that it is more specific and provides additional information by labeling both self-autonomous and phagocytic phases of apoptosis.

From the nanotechnology perspective, the sensor exemplifies a bio enabled approach that adapts actual biological structures and architectures to the construction of non-toxic molecular scale devices. Generally, individuals new to this method might struggle because its success depends on using a highly active Toro SOM preparation and the correct processing of sections to avoid over digestion by proteinase K.The tissue sections for this procedure should be prepared prior to the assembly of the molecular device. Five to six micron thick sections cut from paraldehyde.

Fixed paraffin embedded tissue blocks are recommended. Place the slide with the tissue section in a slide rack and DW wax in xylene for 15 minutes. After 15 minutes, transfer to a fresh xylene bath for an additional five minutes.

Rehydrate the section by passing the slide through these graded ethanol concentrations. 96%ethanol two times for five minutes each. 80%ethanol for five minutes, followed by water.

Two times for five minutes each. After rehydration digest the section with proteinase K.Using 100 microliters of a 50 microgram per milliliter solution per section incubate for 15 minutes at room temperature in a humidified chamber. Depending on the tissue type, the time of digest may need to be adjusted, but be careful to avoid over digestion as it causes signal disappearance and section disruption when the digestion is complete.

Rinse the slide in distilled water twice for 10 minutes each. Apply 2%BSA for pre blocking incubate for 15 minutes at room temperature in a humidified chamber. During this time, assemble the molecular machine as shown next in this video.

All reagents for assembly of the molecular machine are scaled for a total volume of 25 microliters, which is sufficient for a single detection in an average size tissue section. The volume can be scaled up as needed in a small plastic tube. Combine the following reagents in this order by distilled water.

15%polyethylene glycol 8, 000 T four DNA Ligase buffer, dual labeled oligonucleotide vaccinia topoisomerase one and T four DNA Ligase mixed gently by pipetting. The molecular construct will almost instantly self-assemble in this solution and can be used immediately at room temperature. To begin the procedure for labeling DNA breaks, aspirate the pre blocking solution from the slide.

Apply 25 microliters of the full reaction mix to each section. Place a plastic cover slip over the section and place the slide into a humidified chamber. Protect from light by wrapping the chamber with aluminum foil and incubate for one hour at room temperature.

Although both ligase and topo signals can be observed after the one hour incubation, in many instances, the ligase signal can be further enhanced. To enhance the signal, reapply the reaction mix without vac topo and oligonucleotide one, but with oligonucleotide three and T four DNA Ligase, place a plastic cover slip on the section and incubate in a humidified chamber for 18 hours. At room temperature on the following day, gently immerse the slide vertically in a coplan jar containing water at room temperature to remove the cover slip.

Then wash the section in distilled water three times for 10 minutes each. Rinse the section with sodium bicarbonate buffer. This alkaline solution rinse enhances fitzy fluorescence, which is pH sensitive and is significantly reduced below a pH of seven.

Finally, cover the section with an anti fading solution that also contains DPI for counters, staining nuclei, and apply a cover slip. The sections are now ready for fluorescent microscopy. Shown in this figure is a tissue section of dexamethasone treated thymus where the molecular machine has detected DNAs one and DNAs two type blunt ended DNA, breaks in the thymic cortical areas undergoing apoptosis, green cytoplasmic fluorescence indicating DNA's two type breaks, Marx cortical macrophages digesting nuclear material of apoptotic thymocytes.

This signal is produced by vac topo and localizes to FGA lysosomes with DNA containing five prime hydroxyl. Double strand breaks red fluorescence indicating DNA's one type breaks labels, nuclei of apoptotic thymocytes not engulfed by macrophages. Massive numbers of thymocytes simultaneously undergo apoptosis.

Accompanied by generation of five prime phosphate doublet strand breaks visualized by ligase based labeling. These breaks are located at the nuclear periphery and form ring shaped patterns. All cellular nuclei are in blue visualized by counter staining with dpi.

While attempting this procedure, it's important to remember never to allow the sections to become dry in between additions of different solutions. Also keep all enzymatic preparations on ice prior to adding sections and protect your fluorescent probes from light during the labeling reaction After its development. The technique permitted visualization of all phases of heto cell disassembly.

This sensor itself is an example of the green nanotech approach to the design of nanoscale particles, probes, and machines. This approach combines artificial components with naturally occurring molecular machine parts. The goal is to eliminate toxicity and adverse biological reactivity of these highly dispersed nano materials.