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

