Biomod/2012/DNA Maestros:Results

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Validation of the reporter assay for strand displacement on the Icosahedron

Fig 4. Relief of self-quenching of FAM labels in VU₅^FAM upon annealing with VL₅: (a) Schematic showing the basis of the strand displacement reporter assay. (b & c) Change in FAM fluorescence from VU₅^FAM upon incubation with equimolar amounts of VL₅ at b) 37°C and c) 4°C. The data is presented as normalized values with respect to free TMR dye added as an internal control. Data presented is mean of two independent experiments with standard deviations as error bars.

Self quenching of fluorophores occurs when two or more fluorophores come closer than a specific distance^[1]. A simple situation where self quenching is observed is when the concentration of the fluorescent molecules in solution is high. The proposed mechanism for such an observation is the formation of an intermolecular ground state dimer complex^[2], which makes one of the fluorophores in the complex act as a donor and the other as an acceptor. The greater the spectral overlap between the excitation and the emission spectrum of a fluorophore, the higher is the observed self quenching, as seen in cases of homo-FRET^[3]. Fluorescein has been extensively studied for concentration dependent self-quenching^[4] and we wanted to use such a self-quenching assay as a reporter for strand displacement if it occurred on the DNA icosahedron.

We wanted to use the property that VU₅ has 10 identical overhangs, all within a small volume of ~500 nm³. Thus, if multiple FAM (Fluorescein) labelled 10-mer oligomers (FAM-10-mer) were to be hybridized to VU₅, this would result in an increased local concentration of the FAM label, creating a self-quenched state for the FAM fluorescence. If one were to now somehow displace the ten FAM-10-mer molecules hybridized to each VU₅, the FAM-10-mer molecules would be released in bulk solution which would increase the intermolecular distance between the FAM labels and thereby result in increased fluorescence.

In order to see if this assay worked, we created a complex of VU₅ hybridized to ten FAM-10-mer molecules, called VU₅^FAM (Figure 4a). We first decided to displace the ten FAM-10-mer oligos on VU5FAM by introducing the lower half icosahedron (VL₅) whose overhangs are complementary to the upper half icosahedron, VU5FAM in the solution containing 100 nM added TMR as an internal control. We incubated VL₅ with VU₅^FAM at 37°C. Due to the cooperative nature of complex formation by 1:1 VL₅ and VU₅^FAM the major product of the reaction is the full icosahedron^[5]. Importantly we observed an increase in the fluorescence intensity of the FAM label upon complexation with VL5 (Figure 4b). Importantly, the intensity of the internal control, 100 nM TMR, remains unchanged whether VU₅^FAM was complexed to VL₅ or not. This increase in fluorescence intensity due to the formation of complete icosahedron confirmed that the displacement of the ten FAM-10-mer oligos was indeed successful. More importantly, we see that the percentage of fluorescence quenching that occurred due to the hybridization of the FAM-10-mer oligos on to VU₅ was 38%. Given that the R0 of Fluorescein is 4.2 nm^[6], this suggests that the distance between adjacent FAM oligos onVU5FAM is 4.6 nm. The theoretically expected distance as obtained from a model of the icosahedron currently being built in our host laboratory, turns out to be 4.7 ± 3 nm. The same experiment was also conducted at 4°C to minimize possible contributions to the fluorescence due to temperature-induced dissociation of FAM-10-mer oligos from VU₅^FAM (Figure 4c). These relative changes in fluorescence intensities obtained at 37°C and 4°C are similar, confirming that the contribution to fluorescence intensity due to any pre-melted FAM oligos is negligible.

Aptamer recognition of cyclic-di-GMP causes strand displacement on VU₅^apt

Fig 5: Strategy for making icosahedron sensitive to small molecule cdGMP: (a) The incorporation of cdGMP RNA aptamer (blue strand) into the design of the icosahedron. For the sake of clarity the modification on only one out of the total of ten overhangs is shown. (b) Schematic depicting the change in conformation of the cdGMP aptamer upon binding of cdGMP. (c) Structure of the ligand bound cdGMP aptamer[7].

Fig 6. Strand displacement on half-icosahedron driven by cdGMP binding to RNA aptamer: (a) Schematic of cdGMP binding to an engineered half icosahedron eVU₅^FAM that causes displacement of ten pre-hybridized fluorescent DNA strands. (b) Relief of self-quenching of pre-hybridized FAM labels on eVU₅^FAM upon addition of 100 μM cdGMP. Samples were excited at 488 nm and 547 nm and intensities recorded at 515 nm and 596 nm for FAM and TMR labels respectively. FAM intensities of samples were normalized to TMR intensities. The experiment was done at 37°C. Data presented is the mean of three independent experiments. Error bars indicate standard deviations.

In order to create an icosahedron that could be split into two halves by the additon of cdGMP, we had to first make an icosahedron where each of the ten links that hold VU₅ and VL₅ could dissociate upon addition of cdGMP. In order to achieve this, we created an upper half icosahedron VU₅^apt that was modified at each of its ten single stranded overhangs with the cdGMP aptamer. We modified constituent oligonucleotides U2 and U3 of the Uapt 5WJ such that the 10 bases at their 5´ end are complementary to the 10 bases at the 3´ end of the cdGMP aptamer (Figure 5).

The cdGMP aptamer is actually part of a riboswitch where it is embedded in a longer sequence that forms an expression platform^[8]. Although the conformational rearrangement is well documented, and structures of the free and cdGMP bound states of the cdGMP aptamer are well characterized, whether the 3’ end can actually bring about strand displacement is unknown. In order to test this hypothesis we designed an experiment similar to that described in Figure 4. Here, the ten overhangs of the upper half icosahedron modified with the RNA aptamer (VU₅^apt), are also hybridized to an analogous 5' FAM functionalized 10 bp strand (FAM-10-mer-b) to give eVU₅^FAM. In the presence of 100 μM cdGMP, the ten aptamer domains on eVU₅^FAM would undergo a conformational change. If this conformational change resulted in the displacement of the ten FAM-10-mer-b molecules on eVU₅^FAM, due to increase in intermolecular distance between solution-dispersed FAM labels, we should observe. an increased in the fluorescence intensity of FAM. Importantly we observe that this is indeed the case, with the fluorescence intensity increasing nearly three-fold relative change in the fluorescence intensity upon cdGMP addition. This also suggests that the local environment provided by the cdGMP aptamer in eVU₅^FAM could further quench the FAM labels on hybridized FAM-10-mer-b molecules in addition to the self-quenching effect.

Cyclic-di-GMP triggered release of encapsulated cargo from a DNA icosahedron mediated by strand displacement

Fig 7. Release of encapsulated FD10 cargo from eIFD10 mediated by cdGMP: (a) Schematic of cargo release from the icosahedron eIFD10 engineered with ten cdGMP aptamers. For clarity, only two aptamers are shown. eI_FD10 incorporates both a fixed number and fixed location of acceptor fluorophores Alexa 647, on the scaffold. FD10 molecules were encapsulated within these icosahedra using published protocols (9). (b) Ratio of Donor (D) to acceptor (A) intensities on eI_FD10^5AX before and after addition of 100 μM cdGMP at 37°C. (c) Ratio of Donor (D) to acceptor (A) intensities on eI_FD10^11AX before and after addition of 100 μM cdGMP at 37°C. Samples were excited at 488 nm and the intensities at 515 nm (D) and 670 nm (A) were recorded. Data presented is the mean of three independent experiments with standard deviations as error bars.

Encapsulation of cargo within a DNA icosahedron was done by assembling the engineered, small-molecule sensitive icosahedron in the presence of excess of cargo molecules. Choosing a fluorescently labelled cargo for encapsulation allowed the monitoring of the release process. In this case, FITC-Dextran of molecular weight 10 kDa (FD10) was encapsulated within the icosahedron and the cargo-loaded icosahedra were purified from free FD10 by adapting a previously published protocol that is also described in detail here^[9]. The encapsulated FD10 can act as donor fluorophores in a FRET experiment if there exists proximal acceptor fluorophores, say on the body of the DNA icosahedron. Fluorescein and Alexa 647 is a well-documented FRET pair^[10]. Therefore the aptamer-modified, FD10 loaded, icosahedral DNA scaffold was also functionalized with five Alexa 647 fluorophores at well-defined locations to give eI_FD10^5AX.

If the encapsulated FD10 undergoes a resonance energy transfer with Alexa-647 fluorophores, then the release of FD10 molecules into the bulk solution upon addition of cdGMP, should cause an increase in fluorescence of the FAM labels on FD10. It has been shown that, for this particular polyhedron and FD10, encapsulation results in an average of two FD10 molecules per DNA icosahedron[9]. According to the manufacturer’s specifications, on average one FD10 molecule carries 2-3 FAM molecules. Thus the system has 5-6 donor molecules and 5 acceptor molecules which make it suitable for FRET. When fluorescence of eI_FD10^5AX was recorded in absence and presence of 100 µM cdGMP, it was observed addition of cdGMP caused a significant enhancement of the Donor to Acceptor Fluorescence intensity (D/A) ratio (Fig 7). This is consistent with the decrease in donor fluorescence on FD10 molecules due to their proximity to the Alexa 647 functionalized DNA icosahedron, that is subsequently lost due to their release into the bulk solution upon addition of cdGMP. This resultant loss of FRET is reflected in the gain of donor fluorescence. This was recapitulated on a completely distinct FD10- loaded, cdGMP sensitive DNA icosahedron carrying eleven Alexa fluorophores on the DNA scaffold, namely eI_FD10^11AX.

This indicates that addition of cyclic di GMP, that binds to the RNA aptamer of eI FD10 complexes causes a conformational change that results in a strand displacement reaction that displaces the cognate lower half of the icosahedron from its engineered upper half. This splits the icosahedron into its two constituent halves that in turn releases its encapsulated cargo.

References

1. Zhuang, X., Ha, T., Kim, H. D., Centner, T., Labeit, S. & Chu, S. Fluorescence quenching: A tool for single-molecule protein-folding study. Proc. Natl. Acad. Sci. USA 97, 14241-14244 (2000)

2. Kim, T. W., Park, J. H. & Hong, J. I. Self-quenching Mechanism: the Influence of Quencher and Spacer on Quencher-fluorescein Probes. Bull. Korean Chem. Soc. 28, 1221-1224 (2007)

3. Gautier, I., Tramier, M., Durieux, C., Coppey, J., Pansu, R.B., Nicolas, J.-C., Kemnitz, K. & Coppey-Moisan, M. Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins. Biophys. J. 80, 3000–3008 (2001)

4. Lakowicz, J. R. in Principles of Fluorescence Spectroscopy, Vol 1, Third Ed., Springer, p 69.

5. Bhatia, D., Mehtab, S., Krishnan, R., Indi, S.S., Basu, A. & Krishnan, Y. Icosahedral DNA nanocapsules via modular assembly. Angew. Chem. Int. Ed. 48, 4134-4137 (2009).

6. A. Kawski, Excitation energy transfer and its manifestation in isotropic media, Photochem. Photobiol. 38, 487–508 (1983)

7. Smith, K. D., Lipchock, S. V., Ames, T. D., Wang, J., Breaker, R. R. & Strobel S. A. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat. Struct. Mol. Biol. 16, 1218-1223

8. Sudarsan, N., Lee, E. R., Weinberg, Z., Moy, R.H., Kim, J. N. , Link, K. H. & Breaker R. R. "Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321 411–3 (2008).

9. Bhatia, D., Surana, S., Chakraborty, S., Koushika, S.P. & Krishnan, Y. A synthetic, icosahedral DNA-based host-cargo complex for functional in vivo imaging. Nat. Commun., 2, 339 (2011)

10. Bradshaw, J. M., Kwan, J. K., Ling, A. E. H. & Shaw, D. FRET-based binding assay. Apr, http://www.freepatentsonline.com/8153390.html (2012)

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