Stacking nonenzymatic circuits for high signal gain

Abstract

Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toeholdmediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.amplifier | enzyme-free | DNA circuitry S ignal amplification is a ubiquitous theme in biology and engineering, and the ability to amplify signals at the molecular level in large measure determines the complexity and robustness of the molecular devices and systems that can be built. Over the past decade, DNA has been established as the ultimate "intelligent material" to build complex structures, circuits, and devices. DNA circuits have been integrated to form complex Boolean networks (1) and molecular neural networks (2). Such programmed circuits have begun to have applications in ordered chemical synthesis (3, 4), multiplexed labeling of biomolecules for fluorescent microscopy (5, 6), and detection of both nucleic acid and nonnucleic acid analytes (7-9). The combination of DNA circuitry and DNA nanotechnology (10, 11) has given rise to DNA robotics (12) and assembly lines (13).The signal amplifiers underlying many of these advances are metastable DNA substrates whose conformational transformations can be catalytically triggered by strand displacement. The first amplifier of this class was designed by Turberfield et al. (14) and was later modified by Seelig et al. by using metastable kissingloop structures (15). Since then, many hybridization-based catalytic systems have been developed, including ones based on topologically constrained interactions (16), entropy-driven strand exchange (17), and catalyzed hairpin assembly (CHA) (18). Some of these schemes allow cascading of catalysis wherein the product of one reaction serves as the catalyst of another reaction. Autocatalytic reactions (17, 19) and cross-catalytic reactions (18) may also be constructed and programmed. Such molecular amplifiers can guard against signal damping during serial signal transductions in nucleic acid circuits and are easily adapted to the integration of logical operations (20).Whereas the outcome of these amplifiers, the ampl...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

“…Synthetic DNA contains a variety of errors, in particular deletions that result in oligonucleotides of length n-1, and such side products are typically not completely removed in PAGE or high-performance liquid chromatography (HPLC) purification procedures. Similar improvements to the ones reported here were also observed in a previous study of a catalyzed hairpin amplifier that used DNA derived from biological sources 21 .…”

“…Multiple such reactions can be chained together into multi-step reaction cascades with a high degree of control over the order and timing of individual reaction steps 3 . DNA strand displacement cascades have been used to create digital and analog molecular circuits [4][5][6][7] , switchable nanostructures [8][9][10] , autonomous molecular motors [11][12][13][14][15] , and non-covalent catalytic amplifiers 13,[16][17][18][19][20][21] . Moreover, DNA devices using strand displacement reactions can be simulated and designed for diverse applications using computer-assisted design software [22][23][24] .…”

“…Large amounts of DNA can be generated by replication in bacteria and the intrinsic proofreading abilities of living systems ensure the purity of the resulting DNA. In fact, several recent papers have recognized the potential utility of biological DNA for nanotechnology applications 21,[26][27][28] . However, the fully double-stranded nature of plasmid DNA has so far prohibited its use as a material for making dynamic DNA devices, which typically consist of multiple oligonucleotides and contain both double-stranded and single-stranded domains.…”

A self-regulating biomolecular comparator for processing oscillatory signals

“…A growing set of tools for engineering in vivo RNA-based logic could be used to design these mechanisms [30][31][32][33]. In vitro, transcriptional circuits [13,34,35] or strand displacement circuits [9,36] could potentially also implement the mechanisms we describe.…”

Protease-based synthetic sensing and signal amplification

“…Engineering synthetic signaling systems have predominantly been realized with gene expression circuits that relay molecular queues either through transcription factors or functional nucleic acids (4)(5)(6)(7)(8). Here, rational engineering strategies are supported by the modular organization and function of transcription factors and regulatory DNA elements (4), as well as the predictability of base pairing interactions (8).…”