Fig 1.
The micro-fluorescence setup used to detect signals from the molecular beacon (MB) probes during hybridization.
(1) Excitation source: 532-nm laser. (2) Adjustment mirror. (3) Excitation beam. (4) Fiber coupler. (5) Optical fiber, the same fiber used to excite (3) the MB and for the collection (10) of emitted by MB light. (6) Working volume of the liquid (7), with target strands (8) that hybridize to the MB (9) thus causing it to emit light (10). The emitted light passes through the 90:10 beam splitter (11), fluorescence filter (12), fiber coupler (13) which couples the light in the fiber (14). The fluorescence is finally measured by a spectrometer (15).
Fig 2.
Fluorescence signals detected using the micro-fluorescence setup.
(a) Molecular beacon (MB) fluorescence spectra acquired in buffers LB1, LB2, Tris-HCl and HEPES. The spectra were acquired without (solid lines) and with (dashed lines) the complementary strand (CS) in the buffer. Inset shows differences in the normalized intensities of the spectra with and without CS in buffers LB1 and Tris-HCl, the black line shape indicates that normalized spectra measured in presence of CS is red shifted. (b) Number of MB copies yielding fluorescence in buffers without CS (open bars) and 60 min after the addition of CS (black bars) for the respective spectra shown on the panel a).
Fig 3.
Characteristics of the fluorescence peaks.
(a) Peak fluorescence signals measured for molecular beacons (MBs) diluted in four different buffers (identified in the key). Open dots correspond to the MB state without a complementary target, and closed dots represent the state with a complementary target. The first measurement was taken less 1 min after adding the target. (b, c) Comparison of buffers to identify components responsible for the unique behavior of MBs in buffer LB1. (b) Fluorescence peak amplitude and (c) fluorescence peak position for MBs with and without the complementary target strand (added ~15 min after the beginning of sample analysis). Each data point is accompanied by its corresponding standard deviation bars.
Fig 4.
Fluorescence peak position as a function of Mg2+ and Na+ concentrations in buffer LB1.
(a) Fluorescence peak position as a function of the Mg2+ concentration. (b) Fluorescence peak position as a function of the Na+ concentration. Open black diamonds show the fluorescence peak position in LB1 without a target strand. Open violet dots show the peak following the addition of the target strand. Black solid line shows the theoretical dependence of the MB melting (unfolding) energy on the concentration of Mg2+ and Na+. Red dashed line shows the expected MB melting energy. Black dashed line shows the expected MB/target duplex energy. Black dashed arrows show transitions expected from the theory and red dashed arrows show those observed in the experiment. E′U and EU are the theoretical and empirical MB unfolding energies. ΔE′F and ΔEF are the theoretical and empirical shifts in fluorescence energy due to hybridization. EH is the gain in MB free energy due to hybridization and E0 is the arbitrary MB free energy in the liquid. (c) Energy diagram of MB/target duplexes in HEPES (black line) or LB1 (red line). The energy of the unfolded MB was set to zero and other states are shown relative to this. The relative height of the energy barriers is estimated from the MB and MB/target duplex melting temperatures. Dotted arrows show structural transformations of the MB during hybridization.
Fig 5.
Time course of fluorescence peak amplitude (a) and peak position (b) of the molecular beacon in LB1. Immediately after the second measurement (point) cell lysate with (closed points) or without (open points) complementary target strand was added. Each data point is accompanied by its corresponding standard deviation bars.
Fig 6.
Fluorescence peak position for the molecular beacon (MB) in buffer LB1.
Measurements are shown before (first two points) and after (third point onwards) the formation of a duplex with a perfectly matching target (PM, black) and a target containing a single mismatch (MM, red). The five base pairs surrounding the position of the MM are shown for the PM and MM targets on the right, with green representing hydrogen bonds and red a missing hydrogen bond. ΔΔG is the free energy difference between the duplexes formed with the PM and MM strands. CS- complementary strand and CS SNM—complementary strand with single nucleotide mismatch.