Sequence Design
Design Process
After having designed our mechanism at the domain level, we needed to fill in our domains with actual sequences. To start, we picked specific lengths for each of our domains. We decided to stick with the general precedent that toeholds be 6 nucleotides in length, then decided that any domain that was part of a probe be 20 nucleotides in length, as this length is both not wastefully long and long enough to ensure that no spontaneous dissociation would occur. We then decided that any domain longer than a toehold, but not part of the probe system should be 15 base pairs in length, as this would not spontaneously dissociate, but could be easily strand displaced.
After these numbers were decided, we consulted a list of 20 nucleotide sequences that are known to be relatively inert and chose sequences from this list to fill in our 20 and 15 nucleotide domains. We used NUPACK and a little trial and error to find our 7 necessary relatively inert toeholds. We then ran a number of simulations in NUPACK to check for unwanted secondary structures and interactions in our designed sequences.
List of Domains
Probe Domains
The length of these sequences is 20 nucleotides.
Domain Name

Sequence (5' → 3')

Complement Sequence (5' → 3')

P_{tr1}

CCAACTCAACCCATTTCATC

GATGAAATGGGTTGAGTTGG

P_{tr2}

TACATACACCAACCTCCACC

GGTGGAGGTTGGTGTATGTA

lp^{†}

TTTTTTTTTTTTTTTTTTTTTTTTTTT

AAAAAAAAAAAAAAAAAAAAAAAAAAA

up_{ca}

ACCTTACCTCTCCCTAACTT

AAGTTAGGGAGAGGTAAGGT

up_{cg}

ACTAACTCCTACCCACACCT

AGGTGTGGGTAGGAGTTAGT

P_{wg}

CCTCTTTCTTATCACTTCAA

TTGAAGTGATAAGAAAGAGG

† The lp domain is 27 nucleotides long.
Toehold Domains
The length of these sequences is 7 nucleotides.
Domain Name

Sequence (5' → 3')

Complement Sequence (5' → 3')

a_{1}

CTCCTC

GAGGAG

a_{2}

ATCCAC

GTGGAT

a_{2}' ^{†}

ATCC

GGAT

z

AAAAAA

TTTTTT

l

CCATTC

GAATGG

u

ACACAC

GTGTGT

wi

GGTAAG

CTTACC

cgi

ATGTTG

CAACAT

cgd ^{‡}

AGATGA

TCATCT

wgd ^{‡}

AGATGA

TCATCT

† The a_{2}' domain is a subset of the a_{2} domain and is 4 nucleotides long.
‡ Toeholds cgd and wgd are the same. This does not change the function of our system because they are never used in the same solution together.
Other Domains
The length of these sequences is 15 nucleotides.
Domain Name

Sequence (5' → 3')

Complement Sequence (5' → 3')

b

CCTCAAAACTTATCC

GGATAAGTTTTGAGG

x

TATCTCCTTTCTATT

AATAGAAAGGAGATA

List of Strands
A star(*) denotes the complement of a domain.
Strand Name

Shorthand Abbreviation

Domain Composition (5' → 3')

Length

Sequence (5' → 3')

Probe for Track 1

P_{tr1}

p_{tr1}

54

CCAACTCAACCCATTTCATCTT  Staple

Probe for Track 2

P_{tr2}

p_{tr2}

54

TACATACACCAACCTCCACCTT  Staple

Probe for Cargo Attacher

P_{ca}

lp* up_{ca}*

81

Staple  TTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTTAGGGAGAGGTAAGGT

Probe for Cargo Goal_{1}

P_{cg1}

lp* up_{cg}* cgd*

87

Staple  TTAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGTGTGGGTAGGAGTTAGTTCATCT

Probe for Walker Goal

P_{wg}

wgd p_{wg}

60

AGATGACCTCTTTCTTATCACTTCAATT  Staple

Track 1

TR_{1}

p_{tr1}* a_{1}* b* z*

47

GATGAAATGGGTTGAGTTGGGAGGAGGGATAAGTTTTGAGGTTTTTT

Track 2

TR_{2}

p_{tr2}* z* b* a_{2}*

47

GGTGGAGGTTGGTGTATGTATTTTTTGGATAAGTTTTGAGGGTGGAT

Walker

W

l x a_{2} b a_{1}

48

CCATTCTATCTCCTTTCTATTATCCACCCTCAAAACTTATCCCTCCTC

Walker Inhibitor

WI

a_{2}* x* wi*

27

GTGGATAATAGAAAGGAGATACTTACC

Walker Trigger

WT

wi x a_{2}'

25

GGTAAGTATCTCCTTTCTATTATCC

Walker Detacher_{1}

WD_{1}

z b a_{1}

27

AAAAAACCTCAAAACTTATCCCTCCTC

Walker Detacher_{2}

WD_{2}

a_{2} b z

27

ATCCACCCTCAAAACTTATCCAAAAAA

Walker Goal

WG

p_{wg}* a_{1}* b* a_{2}*

47

TTGAAGTGATAAGAAAGAGGGAGGAGGGATAAGTTTTGAGGGTGGAT

Walker Goal Detacher

WGD

p_{wg}* wgd*

26

TTGAAGTGATAAGAAAGAGGTCATCT

Cargo Attacher

CA

x up_{ca}

35

TATCTCCTTTCTATTACCTTACCTCTCCCTAACTT

Sub Cargo Attacher

S_{ca}

lp

27

TTTTTTTTTTTTTTTTTTTTTTTTTTT

Cargo 1

C_{1}

u_{1}* x* l*

27

GTGTGTAATAGAAAGGAGATAGAATGG

Cargo Goal 1

CG_{1}

l x u_{1} up_{cg}

47

CCATTCTATCTCCTTTCTATTACACACACTAACTCCTACCCACACCT

Sub Cargo Goal

S_{cg}

lp

27

TTTTTTTTTTTTTTTTTTTTTTTTTTT

Cargo Goal Inhibitor

CGI

x* cgi*

21

AATAGAAAGGAGATACAACAT

Cargo Goal Trigger

CGT

cgi x

21

ATGTTGTATCTCCTTTCTATT

Cargo Goal Detacher

CGD

cgd up_{cg}

26

AGATGAACTAACTCCTACCCACACCT

