Multi-Arm Junctions for Dynamic DNA Nanotechnology

Abstract

Nonenzymatic catalytic substrates have been engineered using toehold-mediated DNA strand displacement, and their programmable applications range from medical diagnosis to molecular computation. However, the complexity, stability, scalability, and sensitivity of those systems are plagued by network leakage. A novel way to suppress leakage is to increase its energy barrier through four-way branch migration. Presented here, we designed multi-arm junction substrates that simultaneously exploit four-way branch migration, with a high-energy barrier to minimize leakage, and three-way branch migration, with a low-energy barrier to maximize catalysis. Original feed forward, autocatalytic, and cross-catalytic systems have been designed with polynomial and exponential amplification that exhibit the modularity of linear substrates and the stability of hairpin substrates, creating a new phase space for synthetic biologist, biotechnologist, and DNA nanotechnologists to explore. A key insight is that high-performing circuits can be engineered in the absence of intensive purification and/or extensive rounds of design optimization. Without adopting established leakage suppression techniques, the ratio of the catalytic rate constant to the leakage rate constant is more than 2 orders of magnitude greater than state-of-the-art linear and hairpin substrates. Our results demonstrate that multi-arm junctions have great potential to become central building blocks in dynamic DNA nanotechnology.

Effective design principles for leakless strand displacement systems

“…Some implementations of thresholding, which rely on preferentially consuming leaked strands before they are able to interact downstream, require slowing down the competing downstream reactions, thereby slowing the intended pathway (10). Recent work proposed using multiarm junction structures to create multiple high-energy barrier steps to leak (19). However, the desired reaction slows down as well, and leak cannot be systematically reduced to arbitrarily low levels.…”

“…Strand displacement reactions underlie molecular motors (4,6), robots (7,8), logic circuits capable of signal restoration, amplification, digital (9,10) and neural network-like computation (11), and controllers that implement prescribed time-varying dynamics (12,13) based on a systematic theory (14,15). Strand displacement-based molecular amplifiers (16)(17)(18)(19) are not only an essential component for signal restoration in digital circuits but also, useful for diagnostic applications (20,21). The successful implementation of synthetic cell-free systems also leads to additional applications at the interface with biology: nucleic acid-based nanodevices have been delivered in vivo (22) as imaging probes (23) or to perform logical computation (24,25).…”

Single-molecule dynamic DNA junctions for engineering robust molecular switches

“…Design of the TWJ-based DNA molecular switch Multi-way junctions are a widely used structure for constructing DNA-based devices. 15,29 Generally, the highlighted two domains I and II can serve as target recognition and signal transduction, respectively. Target binding on domain I can alter the stability of domain II facilitating downstream reactions (e.g.…”

“…[11][12][13] However, the greatest challenge for a TMSD-based molecular switch is the initiation of the reaction in the absence of an input, known as leakage, contributing to a non-negligible amount of background, which limits the engineering of more specic and sensitive tools. [14][15][16] Efforts have been made to reduce the leakage, such as scalable junction congurations, 17 toeless strategies, 16 and mismatch involved TMSD. 18 It is still desirable to develop a new strategy to reduce leakage and guarantee high sensitivity for engineering highly robust DNA molecular switches.…”

Sub‐Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single‐Molecule FRET

“…[4, 12a] Zero-toehold reactions are important as side reactions (or leakage), which can limit the programmability of complex dynamic DNA networks and circuits that utilize strand displacement. [14,15] Our experiment involved reactingt he dsDNAa nd invader strand at the micromolar level for 24 ha nd then quenching the displacementr eaction by diluting to picomolar concentrations at room temperature, followedb ym easurement at times up to severalh ours later;t he cooling and dilution ensured that the reaction stopped. Since branch migration is believed to occur with as tep time of ca.…”