Difference between revisions of "Part:BBa K4665170"

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Octahedron DNA-origami (3024bp long; 42bp edges)

Biology and Usage

DNA origami is a technique that allows for the fabrication of complex nanostructures through guided DNA folding (Majikes & Liddle, 2021). The process involves designing a long ssDNA molecule, referred to as a scaffold, which acts as a backbone for the assembly. The long strand of ssDNA will then be folded into the wanted format using short ssDNA fragments called staples. Almost any form can be achieved by altering the staple sequences.

For the purpose of our project, Sublimestone, an octahedral 3D lattice form was chosen that acted as a nucleation site for the second module of our project, calcite mineralization.

The staple sequences below were acquired using an algorithm, resulting in the desired octahedral form. The octahedral structure was chosen for its thermodynamic stability, resisting disassembly or rearrangement (Julin et al., 2020). After careful analysis, it was determined that triangular structures have the highest mechanical strength (Tandon, 2021); however, the simplicity of the shape would reduce the surface area needed for nucleation. Hence, the octahedron was chosen as the base of the lattice.

Row	ID	Type	Sequence
1	octa_42nt_A1	vertex	AGGGCGAAATCTCCAGATTTTTTTGAGTGCTCTT
2	octa_42nt_A2	edge	CATTTCTTGATGGCCTCTCAGCATAACCGTCTGGACTCCAACGTCAA
3	octa_42nt_A3	vertex	TTAATTGCGGATCCAATTTTTTTTCTTCATTGGC
4	octa_42nt_A4	edge	GTTATGGATAAACTTGCGAAAGGTTTCTCCAGTGCAGGGAGTGATCT
5	octa_42nt_A5	vertex	ACTTCTTTGCTGCTTGATTTTTTTCCACGTCGAT
6	octa_42nt_A6	edge	ACCTGCGCACACATCTTGGTTCAGAGTTCAACAGCTCGTCATACTCC
7	octa_42nt_A7	vertex	TCGGACTTCATCTTCGGTTTTTTTTGGTGTGCACTTGGGATAATCTCATA
8	octa_42nt_A8	edge	AGGCCCAATGTAGAAACGCAACAGGTGTGG
9	octa_42nt_A9	edge	AGAACACCGTTTTTTTTTTTTTTTTTCTCATAGGTGTACTGGAACTCGCTGGACGAGCTTGCTGGCGCAAACATTCTTCC
10	octa_42nt_A10	vertex	TGCTGCTGAAGTGCCAGTTTTTTTCTGGACCGTATGGGTTGCGCGCTTCTT
11	octa_42nt_A11	edge	TGTGAACTTTGACAGTGTCTTACCGGTGTCACAGACCCCAGTCGTAA
12	octa_42nt_A12	vertex	CAGTTCCAACTTGTGCTTTTTTTAGTCTGTGAA
13	octa_42nt_B1	vertex	CCTTCATCAACCTCCGATTTTTTTGTGCCTTAACGCTCCTGCCTGAGGAG
14	octa_42nt_B2	edge	CCAGCAGCAGTTGAGGACTTAACGTCGATT
15	octa_42nt_B3	edge	ACGCTGCGATTTTTTTTTTTTTTTTTCCACACTTAACGAGGCCAACAAAAGTTGAACTCGTCGAATCCACCCTGCCTGC
16	octa_42nt_B4	vertex	AAGATTCCGCAGCTCGCTTTTTTTAGACGGTCCTGTGGTTCTCCTGTTGAA
17	octa_42nt_B5	edge	TACCCAAGAGTTGGGGGATTCATTGTGATG
18	octa_42nt_B6	edge	CGGTGTTCTTTTTTTTTTTTTTTTTTTGGGAGTTTCTGTCTTCCTCCATAGAGGGCGGAACAGCTGATGGTGTACTTCCT
19	octa_42nt_B7	vertex	TGCATTCTGTTTCTGGTTTTTTTAGAGAACTTTAGCATCTTCCATTGCTT
20	octa_42nt_B8	vertex	GCGCGTAACAGGGCGCGTTTTTTTTCATATGGGGATTCCTTCAGGTTGTC
21	octa_42nt_B9	edge	CCGCTACCACCACAGTGTAGCGGTCACGCT
22	octa_42nt_B10	edge	CGGTGTTCTTTTTTTTTTTTTTTTTTAGAAAGCGAAAGGAGCGGGCGCTAGGGGCGAACGTGGCGAACGATGGTAATGCG
23	octa_42nt_B11	vertex	CTTATAAATCAAATCATTTTTTTAGTTTTTTGGGGTCGAGGCTTATCGGC
24	octa_42nt_B12	vertex	TCGCGGATGTTTCAGGTTTTTTTTCAGCAGCCTTGGCCGAAAGGAAGGGA
25	octa_42nt_C1	edge	TCAGATCGTCACTTCAGTTCCCTGAGTGCT
26	octa_42nt_C2	edge	CGGTGTTCTTTTTTTTTTTTTTTTTTGATCAGTGTGAACACAGCACCCACGAGTGCGGCTCTGGGACGACCAACGTGAGT
27	octa_42nt_C3	vertex	ACAGAATGCATAGTATGTTTTTTTGTGAGTCCTTCAAGGACTCGAGATTGG
28	octa_42nt_C4	edge	CAGTTTCTGGCGGTCAAAGCCTTTTCCTCCAGAGCTTGACAGTCATC
29	octa_42nt_C5	vertex	CTTGACGGGAGCAGGTTTTTTTTCACCGAACAG
30	octa_42nt_C6	vertex	ACTTCTCCCTTCCCAGATTTTTTTTGCTGACCTTGTCTGTACTTCAGGAT
31	octa_42nt_C7	edge	CAGAGAAGCAGTAATGAGTTTCCTTCCAAGGATTCTTGAATGGCACA
32	octa_42nt_C8	vertex	TGTGTTACCAGGATCTTTTTTTTGTGATGGGAA
33	octa_42nt_C9	vertex	CCTGCGTGGCTGCAAATTTTTTTTATTCGTCCAGTTCGGGCCAGATCGTT
34	octa_42nt_C10	edge	ATTGGTTGCGCGATTGTTGAGGTTGTAGTCACAGTGAGTTGCGAATG
35	octa_42nt_C11	vertex	AGTGAGTGGGACTGAATTTTTTTTCACGGATGG
36	octa_42nt_C12	vertex	ATGGGACATTTTCCAGTTTTTTTTCTTGCACAGTGCTGGTTGAGTGTTG
37	octa_42nt_D1	edge	GTGGGCCACTTTTTTTTTTTTTTTTTTCCAGTTTGGAACAAGAGTCCACCCATAGCCCGAGATAGGCCACTACGTGA
38	octa_42nt_D2	edge	ACCATCACCCAAAAGAGAAATCGGCAAAATCC
39	octa_42nt_D3	vertex	GTGATCGGGTCCTCAATTTTTTTACAAGTAGGTCTTAGATGTTAGTATA
40	octa_42nt_D4	edge	TCGCAGCGTTTTTTTTTTTTTTTTTCTGCTCCAATTTGTTCACCAGCTGCTTCGGAAGATGTGTCGTGATCTGTTCG
41	octa_42nt_D5	edge	GCCAAGCACTGGGTCCCCAGAATACCGGACTC
42	octa_42nt_D6	edge	AGAACACCGTTTTTTTTTTTTTTTTTGTTAGCCTCCTCAAACACCTGCTTCTTTGATCTGATTAGGATTTTAATCAGA
43	octa_42nt_D7	edge	GCGTCAAATTGCTTTCGACACAGCTGTTCATA
44	octa_42nt_D8	vertex	CAGCTGCTCAAACAGATTTTTTTCAGAGTTGATACCAATTTCCTTGGTA
45	octa_42nt_D9	edge	GAGGTGAAGTGTAATGAGGTAACACTTGTC
46	octa_42nt_D10	edge	ACGCTGCGATTTTTTTTTTTTTTTTATTTCCTGAACCTCTCAGGACGGTGACCGCTCTTGTTCAGTCATTGAAGATG
47	octa_42nt_D11	edge	GCCACGAGCTGTTGTACAATGTGGCTCTCATT
48	octa_42nt_D12	vertex	AGGGAAGGCATGATGCTTTTTTTTGCGGAAATCTTCAGCAACCCCAAGC
49	octa_42nt_E1	edge	TCTGGTCCAGCCTGAGACGGCGCTCAGCAC
50	octa_42nt_E2	vertex	AGATTCCTCTGCTCTTTTTTTTTTGGGCAGGGCTCCGGTCCTCATAGTC
51	octa_42nt_E3	edge	CACCACCAGCCACTTCAGCTCATTGTCTGG
52	octa_42nt_E4	edge	AACTCTCGAAAGCCAGCCCCCGATTTAGAG
53	octa_42nt_E5	edge	TTCGAGGCTTCATGCGATGTCCTTCCAATC
54	octa_42nt_E6	vertex	CTTGATCAGTAAACCCTTTTTTTACTTTCTCTGAATACTCGTTGAGGCT
55	octa_42nt_E7	edge	GTGGCCCACTTTTTTTTTTTTTTTTAGGGATAACAATCTTATATTTGTCGCTGTTCTCCGCGGACTCGTGCCAATTC
56	octa_42nt_E8	edge	ATCAGAAACTGACAGGGGAACAGATCCAGCCA
57	octa_42nt_E9	edge	GTGGCCCACTTTTTTTTTTTTTTTTCAGCCAGGTTTCGACGTTATTGGATGTTCTTCATCTCCAGGCGTGCTTAGAC
58	octa_42nt_E10	edge	TTCTCATCGCAGGGTCTCACGCACTCCTTCAG
59	octa_42nt_E11	edge	GTGGCCCACTTTTTTTTTTTTTTTTGCGCTGGCATCTCTGCAATTGGTCTCTTCTCCTCCAGGAATGGTCCTTCTTG
60	octa_42nt_E12	edge	ATATCTGTCGCTGCGCCGATGAAGTAGAAGCG
61	octa_42nt_F1	edge	TCGCAGCGTTTTTTTTTTTTTTTTTTCTTCGTAGGTACCTATTAAAGAACGTATCAGGGCGGAATCCCTGCTGGAAC
62	octa_42nt_F2	edge	AGAACACCGTTTTTTTTTTTTTTTTTGCT
63	octa_42nt_F3	edge	GTGGGCCACTTTTTTTTTTTTTTTTACCACTCCCATCCTCAAAGCGGCGCTTACCAGGGACTGATGGGCCCACAATC
64	octa_42nt_F4	edge	ACGCTGCGATTTTTTTTTTTTTTTTATAGCCTGTGTCAAGTCAACCATCTGACCTCAACTCTCCCAGGCCCATCTTC
65	octa_42nt_F5	edge	GTGGGCCACTTTTTTTTTTTTTTTTCTCAGAGCCTGGACCTTGAGGGCTCTGGAGTTCTCTTGACATGGTATTCAGC
66	octa_42nt_F6	edge	CGGTGTTCTTTTTTTTTTTTTTTTTTGTCTGGATCTGCATGATTCTTCCCATACACGAGGATCAATGCGTCCTGGATG
67	octa_42nt_F7	edge	GTGGGCCACTTTTTTTTTTTTTTTTAGGATGACTTCGGCTCCTTTACCTGCAGTTCACCTTGAATCCCTCGGAATAG
68	octa_42nt_F8	edge	TCGCAGCGTTTTTTTTTTTTTTTTTTCGGAACCCTAAAGGGGCGCTGGCAACCCGCCGCGTGCCGTAAAGCACTAAA
69	octa_42nt_F9	edge	GTGGCCCACTTTTTTTTTTTTTTTTTCGCCCACCTGGTTGCCCCACAAATCCACCCTGGGGCAGGAGGCATCTATTG
70	octa_42nt_F10	edge	TCGCAGCGTTTTTTTTTTTTTTTTTGGCAGACCCAGCAACACCTCAGATTGCTTTCCTGACCAGAGATGTGCCGCGA
71	octa_42nt_F11	edge	AGAACACCGTTTTTTTTTTTTTTTTTGAGCTGCGTCCGGTCAAGGGCACCTTACTTCATTGGGTCCACAGCTCCCTGA
72	octa_42nt_F12	edge	ACGCTGCGATTTTTTTTTTTTTTTTAGCAGGTCATCGTCCAGGATGGTCAGAGGTTGTGAACTGGCCCAAGATCTCC

To get the ssDNA sequence a pScaf phagemid with insert for generating DNA origami was used. This phagemid was constructed by Nafisi et al., (2018). They converted pUC18 into a pagemid for custom ssDNA production by adding four components: 1) a full-length M13 origin for ssDNA initiation. 2) Kpnl and BamHl restriction sites for insert cloning. 3) M13 PS for phage particle export. 4) modified M13 origin to serve as the ssDNA synthesis terminator.

Characterisation

Conversion into ssDNA scaffold

This scaffold sequence must first be converted to a ssDNA sequence before being suitable for folding experiments. In our project, we followed the protocol created by Noteborn et al. (2022). First, the dsDNA scaffold sequence was amplified using a standard primer (5' GGGATTCATGGTGTATTGCTTCACC 3') in combination with a modified primer containing 5 phosphorothioate linkages(5'C*A*T*A*T*GACGCGCCCTGTAGC 3') (ordered from IDT). These linkages are introduced at the first 5 basepairs of the 5' end of the desired scaffold strand. After PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease (obtained from New England Biolabs). This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.

Figure 1. Gel of ssDNA scaffold (1) Plasmid containing dsDNA scaffold (2) dsDNA scaffold (3) ssDNA scaffold

Well (1) shows the pScaf_3024 plasmid in which the scaffold sequence is incorporated (acquired from Douglas Laboratories through Addgene). In the second well, the dsDNA scaffold resulting from the PCR using the phosphorothioate primer is observed at ~3000 bp. The third well showcases the dsDNA scaffold sequence from well 2 post-T7 exonuclease digestion. Consequently, well 3 should contain the ssDNA scaffold sequence. A very faint band that is slightly shifted downwards is observed, indicating the presence of the ssDNA counterpart of the scaffold sequence.

Folding Experiments

We assessed the folding of the ssDNA scaffold into our target octahedron using diverse conditions as specified in the procedure by Wagenbauer et al. (2017). Three different factors were varied: temperature, MgCl2 concentrations, and staple-to-scaffold ratios.

Figure 2. (1)T1 (2)T2 (3)T4 (4)T5 (5)T7 (6)T8 (7)RM1 (8)RM2 (9)M5 (10)M10 (11)M15 (12)M20 (13)M25 (14)M30

The prominent bands highlighted within the red boxes represent the surplus of staple strands introduced for the folding experiment. The ~3000 bp fragments correspond to the remaining scaffold ssDNA strands that were not included in the folding process. The bands within the wells, indicated by the green box, and some faint bands positioned above the DNA ladder (arrow) suggest the existence of octahedron structures. Octahedron structures are anticipated to have reduced mobility within the gel, hence they are expected to be situated above the 3024 bp band corresponding to the scaffold sequence.

Transmission Electron Microscopy

Negative-Stain Transmission Electron Microscopy (TEM) was performed to visualize the octahedron structures after the folding experiments. The sample preparation was performed by an experienced technician from the M4I institute in Maastricht. Two continuous carbon grids were used per sample for the TEM sample preparation. The continuous carbon grids were glow discharged for 10 seconds and subsequently 10-15µl of the DNA origami sample was applied. This was incubated for 10 minutes and excess liquid was removed with filter paper. The carbon grids were stained with Uranyl acetate and allowed to air-dry for contrast enhancement.

[Ask Floor nicely to put in the three drawing photos]

The TEM images reveal structured DNA formations, hinting at the existence of octahedral DNA structures. However, to reach a conclusive answer, further measurements and subsequent SEM images for three-dimensional visualization would be required.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 919
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 919
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 738
Illegal BglII site found at 1310
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 919
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 919
Illegal NgoMIV site found at 132
1000
COMPATIBLE WITH RFC[1000]

References

Julin, S., Keller, A., & Linko, V. (2022). Dynamics of DNA Origami Lattices. Bioconjugate Chemistry, 34(1), 18-29. https://doi.org/10.1021/acs.bioconichem.2c00

Majikes, J.M. & Liddle, J.A. (January 8, 2021). DNA Origami Design: A How-To Tutorial. Journal of Research of the National Institute of Standards and Technology, 126:126001. https://doi.org/10.6028/jres.126.001

Nafisi, P. M., Aksel, T., Douglas, S. M. (2018) Construction of a novel phagemid to produce custom DNA origami scaffolds, Synthetic Biology, Volume 3, Issue 1, https://doi.org/10.1093/synbio/ysy015

Noteborn, W. E. M., Abendstein, L., & Sharp, T. H. (2020). One-Pot synthesis of Defined-Length SSDNA for multiscaffold DNA origami. Bioconjugate Chemistry, 32(1), 94–98. https://doi.org/10.1021/acs.bioconjchem.0c00644

Tandon, S. et al. (August 2021). Experimental investigation on tensile properties of the polymer and composite specimens printed in a Triangular pattern. Journal of manufacturing Process, 68A: 706-715. https://doi.org/10.1016/j.jmapro.2021.05.074

Wagenbauer, K. F., Engelhardt, F. a. S., Stahl, E., Hechtl, V. K., Stömmer, P., Seebacher, F., Meregalli, L., Ketterer, P., Gerling, T., & Dietz, H. (2017). How we make DNA origami. ChemBioChem, 18(19), 1873–1885. https://doi.org/10.1002/cbic.201700377

@@ Line 484: / Line 484: @@
 <b> Conversion into ssDNA scaffold </b>
-This scaffold sequence must first be converted to a ssDNA sequence before being suitable for folding experiments. In our project, we followed the protocol created by Noteborn et al. (2022). First, the dsDNA scaffold sequence was amplified using a standard primer (5' GGGATTCATGGTGTATTGCTTCACC 3') in combination with a modified primer containing 5 phosphorothioate linkages(5'C*A*T*A*T*GACGCGCCCTGTAGC 3'), thus introducing these linkages at the first 5 basepairs at the 5' end of the desired scaffold strand (primers were ordered from IDT, * indicating phosphorothioate linkages). After PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease (obtained from New England Biolabs). This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.
+This scaffold sequence must first be converted to a ssDNA sequence before being suitable for folding experiments. In our project, we followed the protocol created by Noteborn et al. (2022). First, the dsDNA scaffold sequence was amplified using a standard primer (5' GGGATTCATGGTGTATTGCTTCACC 3') in combination with a modified primer containing 5 phosphorothioate linkages(5'C*A*T*A*T*GACGCGCCCTGTAGC 3') (ordered from IDT). These linkages are introduced at the first 5 basepairs of the 5' end of the desired scaffold strand. After PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease (obtained from New England Biolabs). This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.
 <html>
@@ Line 508: / Line 508: @@
-The first well shows the pScaf_3024 plasmid in which the scaffold sequence is incorporated (obtained from Douglas laboratories via Addgene). The second well shows the dsDNA scaffold that was obtained after PCR with the phosphorothioate primer. The third well shows the dsDNA scaffold sequence from well 2 after digestion with T7 exonuclease. Well (3) should therefore contain the ssDNA scaffold sequence. A very faint band that is slightly shifted downwards is observed, indicating the presence of the ssDNA counterpart of the scaffold sequence.
+Well (1) shows the pScaf_3024 plasmid in which the scaffold sequence is incorporated (acquired from Douglas Laboratories through Addgene). In the second well, the dsDNA scaffold resulting from the PCR using the phosphorothioate primer is observed at ~3000 bp. The third well showcases the dsDNA scaffold sequence from well 2 post-T7 exonuclease digestion. Consequently, well 3 should contain the ssDNA scaffold sequence. A very faint band that is slightly shifted downwards is observed, indicating the presence of the ssDNA counterpart of the scaffold sequence.
 <b> Folding Experiments </b>
-The folding of the ssDNA scaffold into our desired octahedron was tested in varying conditions in accordance to the protocol created by Wagenbauer et al. (2017) . Three different parameters were tested: temperature, MgCl2 concentrations and staple: scaffold ratios. A gradient PCR was performed with different temperature gradient-ramps. Lastly, different staple to scaffold ratios were tested: 50 nM scaffold : 200 nM staples and 20 nM scaffold : 200 nM staples.
+We assessed the folding of the ssDNA scaffold into our target octahedron using diverse conditions as specified in the procedure by Wagenbauer et al. (2017). Three different factors were varied: temperature, MgCl2 concentrations, and staple-to-scaffold ratios.
 <html>
@@ Line 531: / Line 531: @@
 </img>
 <br>
-<b>Figure 1.</b> The reversible hydration reaction of CO<sub>2</sub> into HCO<sub>3</sub><sup>-</sup>, followed by CaCO<sub>3</sub> formation
+<b>Figure 2.</b> (1)T1 (2)T2 (3)T4 (4)T5 (5)T7 (6)T8 (7)RM1 (8)RM2 (9)M5 (10)M10 (11)M15 (12)M20 (13)M25 (14)M30
      </div>
 </html>
+The prominent bands highlighted within the red boxes represent the surplus of staple strands introduced for the folding experiment. The ~3000 bp fragments correspond to the remaining scaffold ssDNA strands that were not included in the folding process. The bands within the wells, indicated by the green box, and some faint bands positioned above the DNA ladder (arrow) suggest the existence of octahedron structures. Octahedron structures are anticipated to have reduced mobility within the gel, hence they are expected to be situated above the 3024 bp band corresponding to the scaffold sequence.
 <b> Transmission Electron Microscopy </b>
-Negative-Stain Transmission Electron Microscopy (TEM) was performed to visualize the octahedron structures after the folding experiments.
+Negative-Stain Transmission Electron Microscopy (TEM) was performed to visualize the octahedron structures after the folding experiments. The sample preparation was performed by an experienced technician from the M4I institute in Maastricht. Two continuous carbon grids were used per sample for the TEM sample preparation. The continuous carbon grids were glow discharged for 10 seconds and subsequently 10-15µl of the DNA origami sample was applied. This was incubated for 10 minutes and excess liquid was removed with filter paper. The carbon grids were stained with Uranyl acetate and allowed to air-dry for contrast enhancement.
-The sample preparation was performed by an experienced technician from the M4I institute in Maastricht. Two continuous carbon grids were used per sample for the TEM sample preparation. The continuous carbon grids were glow discharged for 10 seconds and subsequently 10-15µl of the DNA origami sample was applied. This was incubated for 10 minutes and excess liquid was removed with filter paper. The carbon grids were stained with Uranyl acetate and allowed to air-dry for contrast enhancement.
+[Ask Floor nicely to put in the three drawing photos]
-[results]
+The TEM images reveal structured DNA formations, hinting at the existence of octahedral DNA structures. However, to reach a conclusive answer, further measurements and subsequent SEM images for three-dimensional visualization would be required.