Results: Difference between revisions
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<title> Results </title> | <title> Results and discussion</title> | ||
<h3>Self-assembly </h3> | <h3>Self-assembly </h3> | ||
<li><A HREF="#RNA/DNA hybrid">RNA/DNA hybrid</A></li> | <li><A HREF="#RNA/DNA hybrid">RNA/DNA hybrid</A></li> | ||
<li><A HREF="#dsRNA structure">dsRNA structure</A></li> | <li><A HREF="#dsRNA structure">dsRNA structure</A></li> | ||
<h3>Luciferase knockdown </h3> | <h3>Luciferase knockdown </h3> | ||
<li><A HREF="#Dual-luciferase-knockdown-assay"> Dual-luciferase-knockdown-assay</A> </li> | <li><A HREF="#Dual-luciferase-knockdown-assay"> Dual-luciferase-knockdown-assay</A> </li> | ||
<h3> | <h3> | ||
Revision as of 10:08, 24 July 2011
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<title> Results and discussion</title>
<h3>Self-assembly </h3> <li><A HREF="#RNA/DNA hybrid">RNA/DNA hybrid</A></li> <li><A HREF="#dsRNA structure">dsRNA structure</A></li>
<h3>Luciferase knockdown </h3>
<li><A HREF="#Dual-luciferase-knockdown-assay"> Dual-luciferase-knockdown-assay</A> </li>
<h3>
<A NAME="RNA/DNA hybrid">RNA/DNA hybrid</A>
</h3>
Initially it was investigated in which molar ratio scaffoldstreng and tie strings even collection is most optimal (Figure 9). There was attempted with the following 5 different molar ratio of scaffold bar and tie strings: 1:2,5, 1:5, 1:10, 1:15, 1:20. In Figure 9 we see that there is a band shift in the last 4 has arisen, after the structure has undergone self-assembly procedure. The self-aggregate structure has a lower mobility than the pure scaffoldstreng, suggesting that there is a omkonformation of the structure. Scaffoldstrengen has a mobility corresponding to ca. 300 bp, which is somewhat lower than the actual length of 370 nt. The lower mobility due to the secondary structure scaffoldstrengen assume because internal base pairing (Figure 10A). Length of scaffoldstrengen has however been verified on a denaturing gel (data not shown). By comparing the amount of staple strands of control in Figure 9, in relation to flow in sheets with self-assembly, it appears that a large quantity of staple strands are consumed in self-assembly procedure, so the band is seen in orbit 6-8, then a collection of scaffoldstreng and tie strings. Also shows the PAGE analysis, although the collection is at all scaffoldstrenge, since in orbit 6-8 not seen any band with the same mobility as the pure scaffoldstreng. From this analysis it appears that a molar ratio between the scaffold and staple strings at 1:10 is the optimal. At the gel would be a sharply defined bands not readily improved by increasing the molar ratio. For this reason, the molar ratio 1:10, was used in subsequent analysis of the structure.
<i>Figure 9: Study of optimal molar ratio between the scaffold and staple strands. 1.5% native agarose with 5 mM MgAc and 0.5 x TBE. Dyed with SYBRsafe, run at 100 V for 120 min. at 4 ⁰ C. BANE1: 100 bp marker. BANE22: Staple strings. BANE3: Scaffold. Course 4: The self-assembly (SA) compared 1:2,5. Course 5: SA in relation 1:5. Course 6: SA over 1:10. Track 7: SA over 1:15. Track 8: SA compared 1:20 </i> The design of RNA / DNA hybrid was emphasized that although the collection takes place in a particular order. First assemble the center square and then assembled the rest so that the structure be closed together. This stepwise self-assembly was attempted pictorial done by making self assembly with an increasing number of staple strands are present (Figure 11). It is only by the addition of the second string last issue (I1), the emergence of a clearly defined band. Here is however a very clear band shift compared to the original scaffold, so it is clear that there has been a form of folds of the structure. By adding the first booklet strict seen no clearly defined bands, which means that the structure does not assume any particular solid structure in these steps. It appears, however, that the vague smear moved up a bit for each book added string, so the structure that is assembled in succession, but it is only by the addition of I1 that the structure assumes a fixed structure. It is unclear smear seen in the first 6 tracks can also be caused by contamination with ribonuclease, so the structure was partially dismantled. In this case, the clearly defined bands in the track 10 and 11 suggest that the overall structure is resistant to degradation by ribonuclease. The conclusion may be drawn from this analysis alone.
<i>Figure 11 Examination of the stepwise self-assembly. 6% native PAGE. 1% TBE, 5 mM MgAc, SYBRsafe staining. Run at 100 V for 120 min at 4 ⁰ C. Courses described in Fig. The marker is loaded in a defective well so it is useless. </i> <h3> <A NAME="dsRNA structure">dsRNA structure</A>
</h3> Preliminary experiments indicated the molar ratio between the scaffold and staple strands that gave the most efficient self-assembly and whether initial treatment with SDS would increase this. The result showed that there was no significant improvement with initial SDS treatment, which therefore was not done in subsequent trials. The optimum molar ratio was found to be 1:10 (data not shown). The analysis of whether there is a folds of the structure after self-assembly procedure is done by native 4% PAGE (Figure 12). Band shift between ranges 2 and 4 in Figure 11 shows that there has been a folds of the structure. At the same gel, tested it on defosforylering of 5'-ends of the scaffold and staple strands affects self assembly. Since there is no significant difference between the bands in track 4 and 5, indicated that while the collection is not affected by this defosforylering.
<i>Figure 12 Self-assembly of dsRNA structure and the influence of defosforylering. 4% Nativ PAGE, 5 mM MgAc 1x TBE. 100 V at 4 ⁰ C for 150 min. Course 1: 100 bp marker. Course 2: Scaffold. Course 3: Staple strings. Course 4: Overall structure. Course 5: phosphatase-treated structure. Marked with arrows seen staple strands at the bottom and top overall structure. </i> The stepwise self-assembly was analyzed by the unwinding of self assembly with an increasing number of staple strands are present (Figure 13). Here is also a clear band shift between the pure scaffold and overall structure. It appears that by stepwise addition of staple strands are also taking a progressively larger band shift. The first shift occurs between track 6 and 7, ie by adding string C, and again between track 7 and 8 by the addition of string I. By adding these two strings changed conformations of the structure may substantially. In orbit 14 and 15 shows respectively. pure scaffoldstreng and tie strings that have undergone self-assembly procedure. Here is no band shift, indicating that the band shift that is otherwise seen in the self-assembly reactions, not due to internal annealing between scaffoldstrenge or medium staple strands. Scaffoldstrengen have mobility as a 300 bp long string, which does not match the actual length of 370 nt. This increased mobility is due to the secondary structure of scaffoldstrengen. The overall structure shows a lower mobility corresponding to about 500 bp. It is from this band shift is not to say whether it actually is the designed structure is assembled. The mobility of this structure may be compared with the mobility of the corresponding structure in the RNA / DNA. This overall structure also has a mobility corresponding to ca. 500 bp. These similar values suggest that it is the same structure are collected in both cases, although the two structures do not have the same design of the booklet strings or sequence of scaffoldstreng. To verify that this is the designed structure is actually assembled, it can be analyzed using different techniques. This can be with small-angle X-ray scattering (SAXS), which can analyze the shape and size of the formed particles. The size would also be determined by dynamic light scattering (DLS) and the particles could be visualized with atomic force microscopy (AFM).
<i>Figure 13 Successive self-assembly of dsRNA structure. 4% PAGE, 1x TBE, 5 mM MgAc. 100 V for 180 min at 4 ⁰ C. Course 1: 100 bp marker. Course 2: Scaffold. Course 3: Staple strings. Course 4: Self-assembly (SA) with booklet string D. Runway 5: SA with staple strands D, A. Course 6: SA with staple strands D, A, B. Course 7: SA with staple strands D, A, B, C. Bane 8: SA with staple strands D, A, B, C, I. Course 9: SA with staple strands D A, B, C, I, C. Bane 10: SA with staple strands D, A, B, C, I, F, E. Bane 11: SA with staple strands D, A, B, C, I, F, E, H. Bane 12: SA with staple strands D, A, B, C, I, F, E, H, J. Bane 13: SA with staple strands D, A, B, C, I, F, E, H, J, G. Bane 14: SA Scaffold. Runway 15: SA booklet strings. Marked with arrows are A: Overall structure, B: Scaffold and C: Booklet strings.</i>
<h3><A NAME="Dual-luciferase-knockdown-assay">Dual-luciferase-knockdown-assay</A></h3>
The purpose of the design of oktahedron structure made of dsRNA, was to make a knock-down effect of Renilla luciferase. To investigate the knockdown effect of our structure was transfected into a stable H1299 lung cancer cell line, after which a dual-luciferase assay was performed.
<embed src="knockdown.jpg" width="100%" height="60" align="center"> <i>Knockdown effect caused by the designed RNA structure studied with dual-luciferase assay. Values are calculated from 3 replicates by transfection with the purified, respectively. uoprensede structure. All control experiments are performed on 4 independent experiments. siEGFP and siEGFP-Cy3 are positive controls with complete base pairing between target and siRNA. siEGFPmis is negative control with no complementary sequence to the EGFP site. "Tranf" are cells exposed only to transfektionsreagens. "Mock" cells are only added RPMI. For each reporter was Renilla luciferase (Rluc) / Firefly luciferase (Fluc) ratio corresponds to "siEGFPmis" control.</i>
The two positive controls gave both a knockdown at around 96% (P = 0.01441 and P = 0.01450), representing significant knock downs. Transfection with the purified structure, a knockdown at around 40% (P = 0.1425), compared to siEGFP mismatch. In cells transfected with a solution containing both the overall structure and tie strings seen a knockdown at around 80% (P = 0.02630) compared to the negative control. The difference in knockdown between the two solutions may be due to the stoichiometric difference in transfection. It was not possible to determine the concentration of the structure of solutions for self-assembly, but the estimated concentration of uoprensede structure transfektionsopløsningen is 16 nM, ie lower than the 40nm as the control solutions contained. The concentration of the purified structure must be lower than 16nM, due to the loss in purification process, which can be give explanation for the lower knock-down effect. The gel analysis confirmed that the structure was present after purification (data not shown). For each knockdown assay, the statistical P-value calculated, respectively. 0.1425 and 0.02630 for the purified and uoprensede structure. Based on these values, there is no significant evidence that the purified structure causes a knockdown, but the uoprensede, however, there is evidence that there is a knock-down effect, if using a P-value for significance at 0.05 . There is therefore a clear basis for further investigation of the knockdown effect. Prior to luciferase assay was performed, cells were examined under light microscope mode. Cells transfected with the purified structure was generally shared well and there were many cells in the wells. Cells transfected with the uoprensede structure had however had it harder, and cell concentration was significantly lower in these wells than in others. This may be due to the many ssRNA staple strands may have started an immune response in cells so that their replication was reduced. The observed luciferase activity in cells transfected with uoprenset structure was therefore also generally lower, giving a larger uncertainty. Since it is the ratio of Firefly luciferase activity and Renilla luciferase activity under investigation data should still be usable.
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