1
Supplementary Data
Detection of a point mutation.
The structures of the transition molecules and the model molecular markers used for
detection of point mutation (Supplementary Fig. S2) are given in Table S8. In the
experiment, each transition molecule was taken at 1 M and total concentration of the
model markers was set to 2 M. The ratio between the sequences was gradually varied
as shown in Supplementary Fig. S3b. The mixture of the transition molecules and the
model markers was equilibrated for 10 min at 15 oC; one-step computation was initiated
by addition of 1 M of FokI enzyme and proceeded at 15 oC for 30 min prior to
quenching and analysis by denaturing PAGE.
Table S8. Molecules used for the detection of the point mutation
Species
Yes → No
transition
Yes → Yes
transition
Structure
TGAAGGAACTGTTACACATGGATG
TGACAATGTGTACCTACGTCC
TGCAGGAACTGTTACACATGGATGTG
TGACAATGTGTACCTACACGTCC
Wild-type
model sequence
3' - ACGTCCTTGACAATGTGTACATCAAC
Mutated model
sequence
3' - ACTTCCTTGACAATGTGTACATCCAC
2
Adjusting confidence in a positive diagnosis for various concentrations of the
molecular indicator.
The experiment described in Supplementary Figs. S3 (c-e) was performed using the
1
 Yes and
SCLC diagnostic molecule. The computation advanced by Yes ASCL
2
 Yes transitions, then branched on INSM1↑ symbol due to regulation by the
Yes GRIA
1

 Yes
INSM ssDNA model disease marker and proceeded to completion via Yes PTTG
1

 No transitions, to reflect the Yes/No ratio obtained at the branching
and No PTTG
point. All transition molecules except the regulated pair and the diagnostic string were
taken at 1 M concentration, and FokI enzyme was taken at 5.4 M concentration. To
improve the regulation pattern, a single-stranded protecting oligonucleotide for the
1

 Yes transition molecule was taken at the same concentration (indicated in
Yes INSM
the Supplementary Fig. S3) as the active regulated transitions.
Release of ssDNA molecule with a sequence identical to the approved antisense
drug
In order to introduce the N-mer ssDNA drug into a diagnostic molecule, we form an (N4)-mer loop due to the restriction pattern of FokI. To design a drug suppressor we use a
complementary ssDNA sequence for the ssDNA drug loops that are equal to or less than
12 nucleotides (N ≤ 16). For longer ssDNA drug loops, this sequence is furnished with
additional nucleotides at its 3'-terminus that are complementary to 5'-region of the
original single stranded loop and thus decrease the size of the loop to 12 nt or less. This
design prevents an active drug from hybridizing to the inactive drug suppressor and vice
versa.
We demonstrate this principle by designing a drug-releasing moiety that administers a
ssDNA molecules that has the same nucleotide sequence as the Vitravene® (FDA
3
approved antisense drug) which is 21 nucleotides long. This drug requires a 17-mer
loop, therefore the drug suppressor-release moiety was designed to have a 8-mer loop
by elongating the drug suppressor to be 28 nt (instead of 21 nt) long. As the drug
molecule did not contain an internal label, we used a labelled drug suppressor as a probe
to indicate drug release.
Supplementary Fig. S5 shows a formation of the suppressed drug upon positive
diagnosis indicating release of an active drug, and the absence of this species upon
negative diagnosis meaning the contrary. A small amount of undesired crosshybridization product between the drug suppressor probe and the unprocessed
diagnostic molecule suggests that the design should be improved further to eliminate
these interactions completely.
To perform the experiment, the diagnostic molecule, containing one symbolic indicator
GSTP1↓, was incubated at a final concentration of 1 M in NEB4 buffer containing 1
M synthetic (28-mer) 32P-labelled drug suppressor. Positive or negative transition for
GSTP1↓ (1 M) and Yes-verification-transitions (2 M) were added. The computation
was initiated by addition of 5.4 M of FokI to a 10 l total volume. After incubating the
mixture for 30 min at 15oC the reactions were quenched with 10 mM EDTA, mixed
with the loading buffer and analyzed by native PAGE (20%). The exact structures of the
molecular species used in the experiments are given in supplementary table S9.
4
Table S9. Molecules used in Vitravene® release experiment.
Species
Structure
Drug suppressor
CGCAAGAAGAAGAGCAAACGCTTCTTCT
probe
Diagnostic molecule
TTGCTCT
GGTGCGCGACGCTCGACGCTCGACGCTCGCGT
T
GCGCTGCGAGCTGCGAGCTGCGAGCGCA
C
GCGTTCTT