Figure A 3-1. Schematic representations of the wireframe structures (large) with gap regions. Both the large designs (A) flat and (B) tube are made up of isosceles triangles, where each edge of the triangle corresponds to a single DNA double helix. For the large structures the base of the triangle is 128 bp long while the legs are 123 bp long. The vertex of the triangles consists of a single-stranded DNA loop domain of two nt. The gap
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regions in the control structures are filled by additional staple-strands represented by red arrows that are annealed during the folding reaction. Schematic representation of one edge of the triangle can be seen in (C). The blue arrows represent the scaffold, while the green arrows represent the staples. The gap in the large structure design can either be 81 or 86 nt long.
Figure A 3-2. AGE analysis of the T4 DNA polymerase assisted gap-filling of the wireframe DNA structures. The protocol for the extension reactions can be referred to in 5.4.2. Lanes marked with the red label “Gap” indicate structures with gap regions, blue label “Ext” = enzymatically filled structures; “p7560” and “p8064” = respective DNA scaf- fold used for the structure assembly; “C” = control structures with staple-filled gaps; “L” = 10 kb plus DNA ladder (Thermo Fisher Scientific).
The product yield was calculated from the ratio of the intensity of the product band and the entire material in a lane excluding the excess staple strands using ImageJ.198 The corresponding data for the gap and extended products for each structure was plotted on a bar-column graph, as seen in the figure. From the graph it can be concluded that the final product yield is not affected by the absence of a large number of staple-strands (33 % for the small design and 66 % for the large design) during the folding step and is in
A.3 Additional figures from chapter 5
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fact better when compared to the control structures where all the staple-strands were provided.
Figure A 3-3. Wide-field tSEM micrographs for the flat small structures. (A) Folded struc- tures with gap regions; (B) Structures filled by T4 DNA polymerase at 25 °C for 3 min, followed by an inactivation step using 2-Mercaptoethanol at 25 °C for 1 min; (C) Control
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structure, without gap regions were folded with additional complementary staples. Scale bars: 100 nm.
Figure A 3-4. Wide-field tSEM micrographs for the tube small structures. (A) Folded structures with gap regions; (B) Structures filled by T4 DNA polymerase at 25°C for 3 min, followed by an inactivation step using 2-Mercaptoethanol at 25 °C for 1 min; (C) Control structure, without gap regions were folded with additional complementary sta- ples. Scale bars: 100 nm.
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Figure A 3-5. Wide-field tSEM micrographs for the flat large structures. (A) Folded struc- tures with gap regions; (B) Structures filled by T4 DNA polymerase at 40 °C for 3 min, followed by an inactivation step using 2-Mercaptoethanol at 25 °C for 1 min; (C) Control structures without gap regions were folded with additional complementary staples. Scale bars: 100 nm.
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Figure A 3-6. Wide-field tSEM micrographs for the tube large structures. (A) Folded struc- tures with gap regions; (B) Structures filled by T4 DNA polymerase at 40 °C for 3 min, followed by an inactivation step using 2-Mercaptoethanol at 25 °C for 1 min; (C) Control structures without gap regions were folded with additional complementary staples. Scale bars: 100 nm.
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Figure A 3-7. Inactivating T4 DNA polymerase with 2-Mercaptoethanol. The optimizations were performed on the flat small structure for different time intervals (1, 3, 10 and 30 min) in buffer recommended by the supplier at 25 °C. To test its inactivation, the polymerase was incubated with 2-Mercaptoethanol for different time intervals (1, 3, 10 and 30 min) prior to the addition of the flat small gap sample. The AGE results (A) show that the inactivation works within 1 min, as the migration of the product band is comparable to the gap control (lane marked with “Gap” in red).
For further optimization, different concentrations of the dNTPs and the T4 DNA polymerase were tested. These optimizations were also performed on flat small structures for 5 min at 25 °C. The extension reaction was inactivated with 2-
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Mercaptoethanol after 5 min of incubation. From the AGE results (B-left) it can be concluded that while keeping the polymerase concentration constant at 3 units in 20 µL reaction, at lower dNTP concentration the polymerase has a higher exonuclease activity that leads to the digestion of the excess staples along with some amounts of the main product. The effect is reduced for dNTP concentrations >50 µM/dNTP, with 500 µM/dNTP being the optimum concentration.
To test the optimal polymerase concentration, five different concentrations were tested (3, 1, 0.3, 0.1 and 0.03 units in a 20 µL reaction). The AGE results (B-left) indicate that a lower concentration of the polymerase reduces the extension efficiency. The minimum concentration is around 1-3 units, with 1 unit being a borderline case. All the conclusions are backed by the PAGE analysis (B-right).
In summary, the optimum dNTP concentration is 500 µM/nt while the optimum T4 DNA polymerase concentration is 3 units in 20 µl reaction. In order to inactivate the polymerase, 1 min of incubation with 2-Mercaptoethanol is sufficient.
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Figure A 3-8. Inactivation efficiency of 2-Mercaptoethanol for all four structures. The ex- tension reactions were performed for 45 min in the supplier’s buffer at 25 °C. After ex- tension, reactions were quenched with 10 % 2-Mercaptoethanol (final) for 10 min at 25 °C.
From the AGE results (A-i) it can be observed that the 2-Mercaptoethanol inactivated product bands (lanes marked with “ext*” in blue), have a slower migration compared to the product bands that were not inactivated by 2-Mercaptoethanol (lanes marked with “ext” in blue). On further analysis with the PAGE gels (A-ii), the inactivated product lanes produce distinct oligonucleotide bands when compared to the non-inactivated product lane that have a smeary background. This behavior was observed across all the struc- tures.
Similar observations were made in the tSEM micrographs (B). The extension reactions that were not inactivated, produce entities that appear to be degraded or malformed. Scale bars: 100 nm
We conclude the inhibition of the 3’->5’ exonuclease activity of the polymerase T4 DNA polymerase with 2-Mercaptoethanol is crucial to obtain correct products.
Note that the PAGE analysis of the large structures flat and tube still produces extra unexpected bands, which are further discussed in Figure A 3-11.
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Figure A 3-9.Optimization of the extension reaction using T4 DNA polymerase. The op- timizations were performed on flat small and flat large structures for different time points (1, 3, 10, 30 and 100 min) in the supplier’s buffers at different temperatures (12, 25 and 37 °C).
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From the AGE results (A-i for flat small and A-ii for flat large), it can be observed that for temperatures 12 and 25 °C the product band migration for both flat small and flat large across all time points are comparable and correspond to the filled structures. While for the extension reactions for both the structures at 37 °C, the excess staple bands in the bottom of the lanes were observed to be fainter compared to the corresponding bands at 12 and 25 °C. Also, in the 100 min time point the product band along with the extra staple band is inexistent. Both these observations for the reactions at 37 °C support the hypothesis that the polymerase is more processive at higher temperatures and has a higher 3’->5’ exonuclease activity that leads to the digestion of the products while some of the remaining forms aggregates (bands in the pockets of the wells).
The PAGE gels (B-i for flat small and B-ii for flat large) show less background smear at higher temperatures. Nevertheless, for the flat large samples, extra unaccounted oligo- nucleotide bands can still be observed, potentially caused by slippage at secondary structures (see Figure A 3-11).
In conclusion, the optimum conditions for T4 DNA polymerase assisted gap-filling of the wireframe structures is: 25 °C extension for 1-10 min followed by 2-Mercaptoethanol inactivation at 25 °C for 1-3 min (AGE and PAGE analysis for this can be referred in C).
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Figure A 3-10. TEM micrographs corresponding to the time and temperature optimiza- tions of the extension reactions with T4 DNA polymerase (detailed discussion in Figure A 3-9). Scale bars = 100 nm.
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Figure A 3-11. Understanding the low gap-filling efficiency of large (flat and tube) struc- tures. The Gibbs free energy of the gap-regions for the two gap designs (small and large) was calculated using the mfold algorithm.105 The results from the simulation were classi- fied into four categories depending on the computed Gibbs free energy value and the observation from the PAGE analysis. A lower (more negative) Gibbs free energy directly corresponded to stronger hairpin regions. The results were plotted on a heat map graphic using k-router108 for respective structures.
At lower temperatures, nearly all the gap regions have a high probability of hairpin for- mation, while at higher temperatures, the probability of hairpin formation is reduced. However, in the large structures, there are still 13 gap regions with a strong high hairpin formation probability at 40 °C. We hypothesize that hairpins with a Gibbs free energy of less than -7 kcal/mol are likely to undergo a replication slippage when extended with the help of T4.
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Figure A 3-12. Fast folding of gap flat small structures followed by fast T4 DNA polymer- ase assisted gap-filling. The flat small structures were folded with gap regions 4 different folding protocols were tested to assess the minimum time needed to anneal and hybrid- ize the domains of the wireframe structures. A-i agarose gel analysis of the isothermal folding of the gap structures. A-ii page gel analysis of the isothermal folding of the gap
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structures. The four different folding protocols can be referred in the table in (A-iii). With respect to the incubation times, protocol A takes ~16 h, protocol B takes ~1 h, protocol C takes ~10 min and protocol D takes ~6 min. Post-folding, the gap flat small structures were extended by T4 DNA polymerase at the optimized conditions (25 °C for 3 min, followed by 2-Mercaptoethanol inactivation at 25 °C for 1 min).
The AGE results suggest that all folding protocols work with equal efficiency and the expected oligonucleotide bands were present in all cases in the PAGE gels (A-ii). Further analysis by AFM does not show significant differences. Scale bars: 500 nm.
In conclusion, the wireframe DNA origami structures with gap regions can be folded within 6 min instead of the standard 16 h and can be extended within 3 min with the polymerase assisted gap-filling protocol.
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