Detection of blunt-ended 5′OH bearing DNA ends in solution using fluorescent molecular machines. (a) The double-hairpin FITC-labeled oligonucleotide 1 (lane 1) was treated with vaccinia topoisomerase (lane 2) for 1 h at 37 °C, resulting in cleavage into two parts: 14-mer and FITC-labeled 23-mer. The cleaved FITC-labeled part of the oligo with attached topoisomerase (23-mer+TOPO–I) did not enter the 20% acrylamide gel and is located in the well (bright green FITC fluorescence); the cleaved unlabeled part of the oligonucleotide 1 (14-mer) is stained by SYBR Green (dark green fluorescence); the unreacted oligonucleotide without attached topoisomerase (37-mer) is FITC-labeled. The addition of the 21-mer acceptor hairpin, with 5′OH blunt-ends (lane 3) results in appearance of the FITC-labeled 44-mer, due to ligation to the 23-mer TOPO-activated oligo. (b) The 38-mer double-hairpin dual-labeled with FITC and rhodamine (lane 1) reacted with vaccinia topoisomerase for 1 hr at 37 °C and was treated with proteinase K (4 μg, 10 min at 37 °C) to remove the enzyme (lane 2), vaccinia topoisomerase cleaved the double-hairpin producing 23-mer FITC-labeled and 15-mer rhodamine-labeled hairpins; the addition of the 21-mer unlabeled acceptor with 5′OH blunt ends prior to proteinase K treatment results in ligation producing the FITC-labeled 44-mer (lane 3). (c) Graded dilutions of a 37-mer oligonucleotide, labeled with a single FITC fluorophore, were spotted onto nylon membrane in 0.3 μL aliquots (spot diameter ~1.3 mm). Images were taken using an Olympus IX-70 microscope and a MicroMax digital videocamera. 1.25 fmol (1.23 × 10⁻¹⁵ mol) of FITC in a spot corresponds to 45 × 10³ FITC molecules/per the surface area occupied by a cell nucleus 0.01 mm in diameter (1 mm grid shown in red).

Light-emitting molecular machine and its usage for DNA damage detection in situ. The machine self-assembles when vaccinia topoisomerase I (TOPO) binds to the double-hairpin oligonucleotide. The machine operation starts when TOPO cleaves the oligonucleotide at the 3′ end of the recognition sequence. As a result the TOPO-activated FITC-labeled portion of the oligonucleotide continuously separates and religates back to the rhodamine-labeled part. When other acceptors, such as blunt-ended DNA breaks with 5′OH are present in tissue section, the FITC labeled part will attach to them. If T4 DNA ligase is present in reaction solution, it will ligate the remaining rhodamine-labeled part to DNA blunt ends with 5′ PO₄. Therefore, both types of DNA breaks are detected simultaneously and directly.

DNA damage detection in tissue sections using molecular machines: 5′OH blunt-ended DNA breaks detection (a) dual detection of 5′OH and 5′PO₄ blunt-ended DNA breaks (b). (a) DNase I and DNase II treated sections. Sections of the normal bovine adrenal were treated with either DNase I, producing 3′OH/5′PO₄ blunt-ended breaks or DNase II, producing 3′ PO₄/5′ OH blunt-ended breaks. The vaccinia topoisomerase I-based molecular machines bearing single fluorophore and T4 DNA ligase selectively labeled only one type of DNA breaks each. Bar = 25 μm. (b) Dexamethasone-treated apoptotic rat thymus. Simultaneous dual detection of two types of cuts in the thymic cortical areas undergoing apoptosis using molecular machines bearing two fluorophores. Cortical macrophages with engulfed nuclear material (green fluorescence) and apoptotic thymocytes (red fluorescence) are labeled. Green cytoplasmic fluorescence reveals lysosomes containing DNA with 5′OH double-strand breaks. Surrounding thymocytes undergo apoptosis and have 5′PO₄ double-strand breaks, located at the periphery of their nuclei (red fluorescence). The lower image was additionally counterstained with DAPI to visualize cell nuclei (blue fluorescence). Red, ligase-based detection; green, topoisomerase-based detection; blue, DAPI staining of cellular DNA. Bar = 15 μm.