is known to slowdown strand displacement rates [126,148,153].
We now recount our journey through multiple sequence designs, focusing on what we learned at each stage and how that informed our design process for subsequent attempts. We would like to stress here that, in particular, our understanding of (i) the challenges described in this sub- section and (ii) our journey through sequence space which is to follow have benefitted greatly from hindsight.
For reasons that will soon become clear, the first and second sequence designs have opposite 5’-3’ orientation for all the strands and multi-stranded complexes relative to later designs and all the domain-level diagrams presented in Chapters3and4.
desired multi-stranded complexes and intermediates were well-formed and that the top strands were (mostly) free of secondary structure.
In our experiments, we encountered two kinds of leak reactions: “initial” and “gradual” leak.
These are illustrated in Figure3.15. Initial leaks involve spontaneous and nearly instantaneous release of the outputs of fuel complexes (such as React and Produce molecules) when a fluorescent reporter for their outputs is present. The fluorescent signal would proceed to completion in as little as 15 minutes.
We found that the initial leak amount scales proportionally with the concentration of the fuel complex but not with the concentration of the reporter for the output (experiments not shown) and this suggested that the initial leak arises due to a fraction of “bad” fuel complexes. At this stage, we did not have a hypothesis for what causes some fraction of the fuel complexes to be “bad”.
For Design 1-PRE, we measured initial leak to be between 8% and 15% for different auxiliary com- plexes. Despite our best efforts at purification of complexes, which included use of PAGE-purified strands, ultramers, modified annealing and gel-purification protocols, we could not reduce this initial leak to much less than 10%.
In contrast to initial leaks, gradual leaks are the slow release of output over the course of the experiment (several hours). They are thought to occur through blunt end strand displacement, or strand displacement which begins by invasion at a junction (see Figure3.12). After preliminary experiments where we observed high gradual leaks, we added 2-nucleotide clamps to the React and Produce molecules (see Figure3.16) to mitigate some of the pathways shown in Figure3.12, such as the React-Produce gradual leak pathway in panel (c). We call this design, augmented with 2-nucleotide clamps, Design 1. The clamps did reduce gradual leak, but not substantially. They did not change initial leak. They are included in Design 1 and all subsequent sequence designs, even if they may sometimes not be indicated in domain level diagrams (for convenience). Sequences that comprise Design 1 are provided in Section5.3.3.
Even with the 2-nucleotide clamps, we observed Produce-Helper gradual leak rates as high as 150 /M /s and React-second input gradual leak rates as high as 50 /M /s in Design 1. A necessary disclaimer about these gradual leak measurements: the leak process that leads to release of output that is measured in these experiments is not truly bimolecular. That is, the gradual leak rate does not scale with the concentration of the reactants exactly as we expect a bimolecular process to. We believe this is because the gradual leak process is likely a conglomeration of multiple pathways,
d
0 5 10
10 20
A leaked (nM)
Time (hours)
Initial leak
Gradual leak
c
Sample 1 Sample 2 Sample 3 Initial: 200 nM Rep A (all samples)
+ ProduceCApAq + ProduceCApAq HelperAAq ___
b
+
+
fA mA sA hAq
f*A mA mA* F
Q sA* sA
mA Q sA sA
mA s*A m*A f*A
fA hAq
F
a
Rep A
f*A mA mA* F
Q sA* sA
s*C
fA mA
sA
f*A
hAp hAp*
hAq sA mA fA f*A hAq*
Produce
CApAqhAq
fA fA
Helper
AAqFigure 3.15: Experiment illustrating leaks in Design 1, withProduceCApAq-HelperAAqleaks as an example. a. The molecules involved. “Rep A”, short for “Reporter for A”, is a molecule used for fluorescence-based readout of the concentration of A using the mechanism shown in panel b. b.
Aq displaces the strand with the quencher (Q), leading to a waste product where the Fluorophore (F) can emit light at its characteristic wavelength. This mechanism works for Ap as well, leading to a quantitative readout of A. c. Experimental setup. All three samples are essentially negative controls for the function ofProduceCApAq, since no FluxCAp is present at any time. Sample 1 shows clean “zero” behavior of Rep A by itself. In Samples 2 and 3, the addition of 100 nM ofProduceCApAq causes an initial release of A. Note that, ifProduceCApAq were to be a perfect molecule, there should be no initial spike since noFluxCAp was present. This “initial leak” is higher in Sample 3 because 50 nM ofHelperAAqwas also added, which suggests thatHelperAAq
facilitates this process. After the initial leak, notice the slower but persistent release of A in Sample 3, which we call the “gradual leak” betweenProduceCApAqandHelperAAq. There is also a much smaller gradual leak between just theProduceCApAqand Rep A in Sample 2.
React
CBCjsC mC fB
s*C
sB mB f*B m*B s*B m*C
f*C
hCj
}
Produce
BCjCks*B
fC mC
sC
f*C hCj
hCj*
hCk sC mC fC
f*C hCk*
}
first 2 nt of mC as a “clamp”first 2 nt of hCj as a “clamp”
a b
Figure 3.16: After preliminary experiments with Design 1-PRE demonstrated very high gradual leak rates, we added 2-nucleotide “clamps” to all React and Produce species as illustrated here, to obtain Design 1. These clamps are meant to mitigate some of the gradual leak pathways shown in Figure3.12, such as the React-Produce gradual leaks in panel (c). The clamps did reduce gradual leak, but not substantially. They are included in Design 1 and all subsequent designs, even if they are not illustrated explicitly in the following diagrams (for convenience).
and we use the bimolecular description as an effective quantitative coarse-graining to describe the process. Taken together, these initial and gradual leaks were too high for our purposes of engineering oscillatory dynamics; we decided to comprehensively revisit our sequence design and verification criteria.