Biomod/2014/fit Results and Discussion.html

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Preliminary Experiment:

verification of FRET with the ssDNAs not modified to the silica

  Fig 7 shows the fluorescence spectra of FITC (50 pM), TAMRA-DNA(50 pM), and their mixture. FITC and Tamra-DNA shows fluorescence maxima at 520 and 580 nm, respectively. The mixture of FITC and TAMRA shows just the sum of the spectra of the two dyes, indicating no FRET occurred.

Fig7.the fluorescence spectra of FITC (50 pM), TAMRA-DNA(50 pM), and their mixture


Fig. 8 shows the fluorescence emission spectra measured by microspectroscopy. We used microspectroscopy because the quantity of the sample is very small. The spectrum of FITC-DNA and TAMRA-DNA showed the peaks at 520 nm and 580 nm, respectively, which is the same result as the ones measured on fluorospectrometor (Fig. 8). When the FITC-DNA and TAMRA-DNA are mixed and hybridized, the fluorescence of the FITC is almost quenched while the fluorescence of TAMRA is enhanced, indicating the FRET induced by hybridization. Thus we successfully detect the hybridization by FRET with microspectroscopy.

Fig8.the fluorescence emission spectra measured by microspectroscopy




 

The synthesis of the Barrel particles and Doll particles

  Fig. 9a and 9b shows the photograph and the SEM image of the amino-functionalized mesoporous silica (MPS-NH2) particle. Spherical particles with the diameter of about 300 nm to 1000 nm are observed. However, because the size of the mesopores is too small, assumed as 2 nm, we could not observed the pores. After the modification with -COOH group (Fig. 9c) and with DNA (Fig. 9d), there were no significant changes in SEM. Therefore, it is understood that the shape of former particle is maintained.

Fig9.the photograph and the SEM image of the amino-functionalized mesoporous silica (MPS-NH2) particle.



We then observed the samples with CLSM. Fig. 10 shows the CLSM images of the barrel particles, that is the MPS attached with FITC-terminated ssDNA. In Fig. 10, Spherical particles of almost the same size as those observed with SEM is observed. In the fluorescence image at 525±50 nm (Fig. 10b), some green rings are observed, while only very weakly fluorescent image is observed at 595±50 nm (Fig. 10c). Since FITC has the maximum fluorescence wavelength of 525 nm, this result indicate that FITC-DNA was successfully attached to the surface of the mesoporous silica particles. In the combined image (Fig. 10d), the green circles of fluorescence appear to surround the particles. It is thought that, because the ring-like fluorescence is observed, fluorescent molecules with DNA is modified only on the surface of the silica particles. If the fluorescent molecule were fixed also in the pore of inside the particle, and the entire particle should have the fluorescence.

Fig10.(a)Photograph and (b)-(d) SEM images of the (a)(b) amino-functionalized mesoporous silica particles (MPS-NH2), (c) caboxylated mesoporous silica particles (MPS-COOH), and (d) DNA-modified mesoporous silica particle.



Pop-up of the doll by the addition of the sword-DNAs

  We examined the pop-up event executed by addition of sword-DNA-1, which is fully complementary with the barrel DNA, to the Barrel-Doll pair under observation by CLSM. Before adding the sword-DNA-1 (Fig. 11a-c), the ring like fluorescent image due to the fluorescence of FITC attached to the barrel particle is observed. After adding the sword (Fig. 11d-f), the fluorescence at 525 nm is quenched while the fluorescence at 585 nm is enhanced. The Movie 1 shows the real-time change upon adding of sword-DNA-1 to the barrel particle. As time passes, the green fluorescence (525 nm) is gradually quenched while orange one (585 nm) is getting stronger.
This result is clear evidence of the pop-up event. Since the fully complementary sword-DNA-1 was added, the DNA hybridized with the ssDNA on the barrel particles, displacing the Doll-particles. Upon this hybridization, the fluorescence of FITC@Barrel (λex = 525 nm) is quenched by TAMRA@Sword (λem = 550 nm, λex = 580 nm) and the fluorescence from TAMRA is observed by FRET mechanism.

Fig11. CLSM images of the barrel-doll particles (a)-(c) before and (d)-(e) after adding the fully complementary sword-DNA-1: (a)(d) fluorescence image (λex=525 nm), (b)(e) fluorescence image (λex = 595 nm), (c)(f) combination image. The wavelength of the excitation laser is 488 nm.


A barrel particle has a doll particle from a result of Fig13 and knows that the place of the point of the doll particle was blown off when sword DNA of the complete complementarity is added

We then examined the dependence of the base sequence on the pop-up event. Instead of the fully complementary Sword-DNA-1, the party mismatched Sword-DNA-2 and -3 were added to the barrel-doll particle. We have expected that the enhancement of the orange fluorescence due to FRET mechanism is less as the mismatch increases. However, as shown in Fig.13, the quench of the FITC fluorescence and the increase of the TAMRA fluorescence was observed similar to the fully matched Sword-DNA-1 system. shows the CLSM images The orange fluorescent color which FITC has appeared a little, and since FRET should not occur, the result expected since added DNA was a mismatch thought that it did not shine a little in orange. It is thought that 1 or 2 mismatches introduced in the Sword-DNA-2 and 3 is not enough to hamper the hybridization.

Conclusions

 ・We successfully mimicked the pop-up pirate on nanoscale by using the mesoporous silica colloid attacheded with ssDNA. ・The synthesis of the Barrel-particle, the Doll-particle, and the Barrel-Doll pair were confirmed by SEM and CLSM observations. ・The Sword-DNA-1 which has fully complementary sequence with the Barrel-DNA displaced the Doll-DNA, resulting in 'pop-up' of the Doll particle from the Barrel particle.



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