Figure 1.
Components of nanoblinker and its mode of operation.
a. Parts of nanoblinker; b. FRET “on” and “off” cycles powered by Brownian motion.
Figure 2.
Detection of blunt-ended 5′OH DNA in model systems using nanoblinkers.
a. PAGE separation of working parts of nanoblinker: Lane 1. 38-mer Core Oligo;Lane 2. Cycling nanoblinkers assembled by combining Core Oligo with VACC TOPO: equilibrium between 15-mer +23-mer (green and red bands) and 38-mer Core Oligo (yellow fluorescence). Solution was treated with proteinase K to strip VACC TOPO (10 µg/µL, 15 min, 37°C). Lane 3. Same as Lane 2, but without treatment by proteinase K. VACC TOPO–23-mer complex did not enter the 20% acrylamide gel and was retained in the well (red fluorescence). Lane 4. Detection of 5′ OH blunt ended DNA ends. Unlabeled 21-mer Test Oligo was added to the cycling nanoblinkers. Detection reaction created new chimeric 44-mer (red fluorescence). Compare with dual labeled Core Oligo in Lane 3 (yellow fluorescence). Note greater intensity of the green band due to the release of the 15-mer hairpin as a result of the detection reaction. b. FRET-based detection of 5′OH DNA ends. Emission spectra of cycling nanoblinkers before (blue curve) and after (green curve) addition of 5′OH DNA ends - Test Oligo (to final concentration 20 pmol/µL). Addition of same amount of 5′PO4 DNA ends (Test Oligo PO4 ) did not change fluorescence (red curve). λExcitation - 488 nm. For Probes and nanoblinker Core Oligo sequences see Materials. c. Image of the tubes with nanoblinkers taken through the objective of a fluorescence microscope before (left tube), and after (right tube) addition of 5′OH DNA ends. λExcitation - 490 nm.
Figure 3.
Detection of DNA breaks added to the nanoblinker system 30 s and 15 min after the initiation of the cycling reaction.
PAGE separation of fluorescent “detection” products of the cycling nanoblinker system. Nonfluorescent cycling nanoblinker system (38-mer-VACC TOPO complex ↔ 15-mer +23-mer-VACC TOPO complex) created by mixing 100 pmoles Core Oligo-2+100 pmoles VACC TOPO. Green and red fluorescent acceptors added at 30 s and 15 min post assembly: Lane 1. 100 pmoles FAM-labeled 15-mer FAM Test Oligo (green) added to the cycling system at 30 s; Lane 2. 100 pmoles TAMRA-labeled 15-mer TAMRA Test Oligo (red) added to the cycling system at 30 s; Lane 3. 100 pmoles FAM-labeled 15-mer FAM Test Oligo (green) +100 pmoles TAMRA-labeled 15-mer TAMRA Test Oligo (red) added to the cycling system at 30 s; Lane 4. 100 pmoles FAM-labeled 15-mer FAM Test Oligo (green) added to the cycling system at 30 s +100 pmoles TAMRA-labeled 15-mer TAMRA Test Oligo (red) added to the cycling system at 15 min; Lane 5. 100 pmoles TAMRA-labeled 15-mer TAMRA Test Oligo (red) added to the cycling system at 30 s +100 pmoles FAM-labeled 15-mer FAM Test Oligo (green) added to the cycling system at 15 min; The reactions were stopped at 30 minutes. Solutions were treated with proteinase K to strip VACC TOPO (10 µg/µL, 15 min, 37°C). Note the 38-mer bands representing the “detection” products. The yellow color in Lane 3 is produced due to superimposition of fluorescence from red and green 15-mers added at the 30 s time-point and “detected” by ligating to 23-mer-VACC TOPO hairpins. Compare with equally intense yellow 38-mer bands in Lanes 4, 5 produced by “detection” of red and green 15-mers mix added separately at 30 s and 15 min time-points.
Figure 4.
Changes in VACC TOPO cleavage activity are followed by changes in t50% detection time.
a. pH-induced changes in the VACC TOPO rate constant for strand cleavage. The first-order rate constants for strand cleavage in the pH range 4.6–9.8 obtained using single-turnover conditions (from 21 with permission). b. pH-induced changes in the time of nanoblinker-based detection of 5′OH DNA breaks (t50%). Detection time t50% - time interval in minutes required to reach a 50% increase in the starting FRET ratio for each individual pH value.
Figure 5.
FRET-based detection of phagocytosis of apoptotic and necrotic cells.
a. Emission spectra of nanoblinkers with macrophages before phagocytosis initiation (blue curve), and 3 min after the addition of macrophages digesting apoptotic (green curve) or necrotic cells (red curve). λExcitation - 488 nm. (Full spectra and graphical representations of all experiments and additional controls see Tables S2–S3). b. FRET ratios (ED/A) of nanoblinkers detecting phagocytosis of apoptotic and necrotic cells by macrophages. Control - macrophages before phagocytosis initiation by addition of apoptotic or necrotic cells. When tested separately before phagocytosis all cell types (macrophages, apoptotic and necrotic U87 cells) produced indistinguishable low intensity signals (see Tables S2–S3). The spectrofluorimetric assessment presented as Mean ± SD for five independent experiments was performed 3 min post addition of nanoblinkers (see Spectrofluorimetric assessment of phagocytosis using nanoblinkers (ED/A = ED 525 nm/EA 580 nm ); λExcitation - 488 nm.
Figure 6.
Fluorescence microscopy of macrophages digesting apoptotic and necrotic cells.
Phagolysosomes in the cytosol of macrophages are the organelles which process apoptotic and necrotic cells and are labeled by the nanoblinker construct (green). Nuclei of macrophages are visualized by fluorescence stain DAPI (blue). Note more active phagocytosis in case of apoptotic cells. λExcitation - 488 nm. FITC excitation D490/40, emission 520/10; DAPI.