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Spider Silk

Spider silks show outstanding mechanical properties like high tensile strength and elasticity. Nevertheless having 1/6 density compared to steel(1.31 g/cm³), spider silk is strong as steel. Also, because of its ductility and malleability, spider silk could be stretched up to five times in length.

People have been tried to produce spider silks artificially for a long time. There were attempts such as producing Biosteel by transplanting spinning apparatus to silkworms and making spider silk from milk by inserting spider genes to cow. Recently one of bio-venture company, Spiber, is developing polymer fibers made by protein from spider silks to micro-organism.

However all these research have some limits.

Spider silks is composed of amorphous segments and crystalline segments. Major amino acid of amorphous segments and crystalline segments are glycine and alanine respectively. Crystalline segments of spider silk are having form of β-sheet structures.

Because DNA, biomolecule composed of repeating sequence including adenine, thymine, guanine, and cytosine, could form a structure through self-assembly between complementary base sequence, DNA is frequently used as material in nanotechnology. We focused on this character of DNA and tried to re-create spider silks in nanoscale by using DNA. This is how our design, DNA silk, started.

DNA Silk

Using DNA, our team synthesize nanofiber named DNA Silk by mimicking characteristic structural feature of spider silk.
This fibril is formed through connection(via self-assembly?) of enzymatically replicated long DNAs and DNA origami bricks respectively corresponding to amorphous and crystalline segments.

Amorphous segment

By using a rolling circle replication(RCR) process, DNA strands are extracted from circular DNA(e.g. plasmid) as a form of single strands.
(Later, some of these strands is used as scafold for DNA origami.)
To make fibril, disarranged strands after replication should be connected properly.
We thought adding short oligomer to arrange strands by increasing stiffness could be a solution.
After this process, DNA origami is started by putting staples.

Crystalline segment

Although crystalline segments of spider silk have unique features according to kinds of spiders and purpose of spider silk, ratio of ordinary spider silk is x:y=2:7. That's why we made DNA brick which had similar ratio(x:y=2:7) using DNA origami.

1. Brick

By designing DNA bricks into triangular prism and using long DNA stand and staples to produce this structure, the basic unit for fibril could be made.

Followed by connecting these units together, we could make silk.

Brick Cadnano

As we can see, triangular prism is made up of three rectangular.

Two of three rectangular of triangular prism are used for connecting with another DNA brick and the last rectangular is used for connecting functional group.

This functional group is the major part of our structure.

2. Connector

DNA origami structure connecting DNA bricks, the most basic segment, into fiber. It has trigonal prism structure like DNA brick and two faces at prism are designed to attach to DNA brick

3. BCC

By designing DNA bricks into hexahedron and using long DNA stand and staples to produce this structure, the basic unit for fibril could be made.

Followed by connecting these units together, we could make silk.

Two of six faces are used for connecting with another DNA brick and another one face is used for connecting functional group.

This functional group is the major part of our structure.

Brick and Connector are connected by staples.

The figures above show where staple, connecting link, placed.

Structure size

Sections marked in red parts are composed of only scaffold, no staple and each is 12nt.

X = 6 helix = 6 × 2.25nm = 13.5nm = 39.70588235 bp

Y = X × (7/2) = 47.25nm = 138.9705882 bp ≒ 139 bp

Y’ = Y + 24nt = 163nt

1. Length of crystalline : Length of amorphous = 1 : 1

Length of an amorphous section = Y = 139 bp

Length of a scaffold that forms a trigonal prism = Y’× the number of helix = 163 nt × 27 = 4401 nt

Length of a scaffold building a unit = Length of crystalline segment + Length of amorphous segment = 4540 nt

2. Length of crystalline : Length of amorphous = 1 : 5

Length of an amorphous section = Y × 5 = 139 × 5 bp = 695 bp

Length of a scaffold that forms a trigonal prism = Y’× the number of helix = 163 nt × 27 = 4401 nt

Len gth of a scaffold building a unit = Length of crystalline segment + Length of amorphous segment = 5096 nt

DNA silk & Spider silk

A strand of spider silk(=thick strand) consists with several cylindrical structure by covering with thin skin. This cylindrical structure(=thin strand) is made by gelation of protein composites. And protein composites that form cylindrical structure is made of crystalline and amorphous segments.
We're-created amorphous and crystalline segments using DNA strands and DNA origami structures respectively.
The Brick made of DNA strands and DNA origami structures corresponds to thin strand of spider silk and a Brick-Connector Complex(BCC) corresponds to a strand of spiders silk.

Process of Making DNA silk

1. Making long DNA single strand

The first step to produce DNA silk is making long DNA single strand. This long stand could be made via RCR process. For this process, we need to prepare single stand which would act as template for RCR. CircLigase - ssDNA Ligase connect both ends of single strand and form circular DNA. Soon, this CircLigase - ssDNA Ligase is separated and primer attach to circular DNA. Then, polymerase attached to circular DNA and primer, move around the template and duplicate complementary single strand to circular DNA. By repeating this process, we could get a long ssDNA which has repeat sequence. This long ssDNA becomes foundation of DNA silk we made. Some parts of this stand used for making amorphous segments and other parts used for making crystalline segments as scaffold.

2. Brick DNA origami

By adding staples to this scaffold, DNA brick in the form of triangular prism is prepared via DNA self-assembly. DNA brick corresponding to crystalline segments of DNA silk has 2:7 ratio which usual β-sheet of spider silk has. Because one single strand is composed of repeat sequence, several bricks would be formed.

3. Connecting DNA silk

To make DNA silk thick enough, several DNA strands need to be connected. For this, the first thing needs to be done is arranging disordered strands in a row. This process could be brought by putting complementary oligomers. Once complementary oligomers stick to DNA strands, strands become stiff and lose its flexibility.

4. Connect DNA origami

For next step: connecting strands, we need to prepare another structure. This DNA origami structure looks similar with DNA brick, but this one isn't connected with DNA strands. We call these individual triangular prisms Connector. Once DNA bricks mixed with Connector, Bricks and Connectors stick to each other and form hexagonal prism made of 3 Bricks and 3 Connectors. We call this structure BBC: Brick-Connector Complex. By this process, we could connect three strands in to one.

5. Giving flexibility

Similar to spider silk has flexibility because of amorphous segments, DNA silk is flexible because of amorphous segments. For giving flexibility back to DNA strand, oligomers attached to amorphous segments for forming pre-fiber need to be detached. To remove oligomers attached to strand partially, DNA strands whose sequences complement to all sequences of oligomers could be used. Because oligomer-DNA strand bond is more stable than oligomer-amorphous segments bond, oligomer-amorphous segments bond would be broken. By this process, oligomers are removed and flexibility of amorphous segments would come back.

Future Study

Unit structure composed of 2 crystalline segments and amorphous segments

M13mp18 used for making circular DNA has 7,249 bases. This means if we could cut the length of crystalline segments and amorphous segment upto 3500nt, we could make 2 types of crystalline segments in one duplication. In other words, 1 unit composed of trigonal prism A, amorphous segment A, trigonal prism B, amorphous segment B is produced. Also, if Connector could connect both trigonal prism A and B, BCC structure having trigonal prism A and B arranged alternatively could be formed.

Property

Changing length of amorphous semgment	Insert partial double-stranded
Longer single stranded DNA means more flexibility. Therefore, by making amorphous segments longer, single stranded DNA could be more flexible.	Single stranded DNA having 150bp is so flexible that it could be tangled. This problem could be solved by increasing stiffness of single stranded DNA through putting oligomers which could form double stranded DNA with single stranded DNA.

Pre-Fiber

In our design, BCC structure corresponds to spider silk.
In future study, we could make another fiber having unique features by using this DNA silk as a pre-fiber.

Functional Group

By connecting functional group with Brick or Connector, DNA silk could capture material reacting to functional group.

• Process of connecting functional group
  

1. Some staples connected to Brick(or Connector) are not complementary to scaffold.

2. Green parts(=functional group) whose sequences are complementary to sequence of black parts are connected to BCC.

3. When materials reacting to functional group are approaching to BCC, they're be captured by functional group.