Kinetically-enhanced DNA detection via multiple-pass exonuclease III-aided target recycling

One of the promising approaches to address the challenge of detecting dilute nucleic acid analytes is exonuclease III-aided target recycling. In this strategy, the target DNA self-assembles with the reactant DNA probes and displays itself as a reactant and product at the same time. This provides an autonomous mechanism to release and reuse the analyte from each round of reactions for repetitive cycles, which amplifies the signal without amplifying the analyte itself. However, for very low amounts of the analyte, it takes a considerably long time before a detectable signal is generated. Thus, in this paper, we report a kinetically-enhanced target recycling strategy by designing two more target recycling sub-reactions that are triggered by the byproducts of the first reaction involving the target analyte. In this manner, concentrations of up to 0.5 pM of target DNA can be detected in 15 minutes.


S1. Sequence of Oligonucleotides
The sequence of the target is the conserved region of the Salmonella typhimurium strains that causes non-typhoidal salmonella.The sequence is adapted from the paper of Patterson, et al. 1 .The other segments are designed accordingly and are pre-determined in silico using Nupack 2 to avoid unnecessary and undesired secondary structures and sequence complementarity among strands that should not have any interaction.The resulting sequences are summarized in the table below.

S2. Optimization of the buffer magnesium ion concentration.
In order to optimize the signal generation especially from the first target recycling reaction involving the triplex probe P o A 1 A 2 , 10 nM of the target (A o ) was added to a mixture of the probe (100 nM) and 1X NEBuffer 1 supplemented with different concentrations of Mg 2+ .Then, 0.2 U/uL of exonuclease III was added.These were prepared in a 96-well plate, and the fluorescence signal was obtained using Varioskan Lux microplate reader.It can be observed that the 50 mM Mg 2+ solution has the highest signal-to-noise ratio.In addition, since the fluorescence signal when only P o A 1 A 2 is present is due to the incomplete hybridization of the quencher strand to the fluorophore strand, it can be inferred that in magnesium concentrations higher than 30 mM, the triplex probe is more stable (compared to that in 20 mM).So in the succeeding experiments, the NEBuffer, which originally contains 10 mM Mg 2+ , was supplemented with enough Mg 2+ (dissolved in 1X TE buffer) to have a final concentration of 50 mM Mg 2+ .

S3
. Temperature-dependence of the stability of the probes.
Despite using an enzyme with an optimal activity at 37 o C, the reaction was done at 25 o C. Firstly for the convenience of point-of-care detection, and second due to the thermodynamic stability of the probes P 1 S and P 2 S. Using Nupack, the reaction yield can be calculated in silico at various temperatures.At 25 o C, the probe P 1 or P 2 and the stopper strand S form a 96% or 93% yield respectively of the corresponding annealed probe P 1 S or P 2 S.But at 37 o C, the yield drops to 76% or 68% respectively (see Figure S3), forming a high amount of free fluorescent-labelled P 1 and P 2 probes in solution, thereby increasing the background signal.

S4. Mathematical relationship of the signal generated over time in a one-, two-, and threepass target recycling system
Figure S3 is a truncated version of the figure 2 in the manuscript, considering only the region after the dip, i.e., the addition of the exonuclease to the reaction.

Figure S3.
Fluorescence signal generated by one-, two-, and three-pass target recycling reactions from the time exo III is added (time = 5 minutes).The r 2 values show relatively linear relationship with a 1.7-and 3.7-fold increase in slope when one or two more target recycling subreactions were added, respectively.
The kinetics of the signal generation can be derived by assuming first-order reaction for the three reactions: (1) And assuming (2) and (3) are two similar reactions we obtain two rate expressions, one for equation (1) and another for equation (2) which will be similar to (3) as follows: (4) (5) For equation (4), since k 1 and the initial concentrations of we can integrate the expression from time = 0 to time = t and obtain: (6) (where Similarly, since k 2 and are constants, and the concentration of at a given time t is given by the same expression as (6), equation 5 can be integrated from time = 0 to time =t and obtain: (7) (where Finally, a combined expression for the three reactions can be summarized as equation 8 and 9 below, where equation 9 simply takes into account two independent subreactions. ( Comparing hypothetical graphs of equations ( 6), (8), and (9) for the one-, two-, and three-pass reactions in Figure S3 below (generated online through http://fooplot.com),resembles the trend in Figure S2.

Figure S2 .
Figure S2.Reaction yield of P1S and P2S at different temperatures.The equilibrium concentrations of the reactants (P 1 or P 2 and S) and the products (P 1 S or P 2 S) are shown at 25 o C and 37 o C. (Initial concentration: P1 = P2 = S = 1 uM).

Figure S4 .
Figure S4.Graphs of hypothetical equations in the form similar to a one-, two-, and threepass multiple target recycling reaction derived.

Table S1 .
Sequences of oligonucleotides