Programming DNA-Based Biomolecular Reaction Networks on Cancer Cell Membranes.

DNA is a highly programmable biomolecule and has been used to construct biological circuits for different purposes. An important development of DNA circuits is to process the information on receptors on cell membranes. In this Communication, we introduce an architecture to program localized DNA-based biomolecular reaction networks on cancer cell membranes. Based on our architecture, various types of reaction networks have been experimentally demonstrated, from simple linear cascades to reaction networks of complex structures. These localized DNA-based reaction networks can be used for medical applications such as cancer cell detection. Compared to prior work on DNA circuits for evaluating cell membrane receptors, the DNA circuits made by our architecture have several major advantages including simpler design, lower leak, lower cost, and higher signal-to-background ratio.

Fast and compact DNA logic circuits based on single-stranded gates using strand-displacing polymerase.

DNA is a reliable biomolecule with which to build molecular computation systems. In particular, DNA logic circuits (diffusion-based) have shown good performance regarding scalability and correctness of computation. However, previous architectures of DNA logic circuits have two limitations. First, the speed of computation is slow, often requiring hours to compute a simple function. Second, the circuits are of high complexity regarding the number of DNA strands. Here, we introduce an architecture of DNA logic circuits based on single-stranded logic gates using strand-displacing DNA polymerase. The logic gates consist of only single DNA strands, which largely reduces leakage reactions and signal restoration steps such that the circuits are improved in regard to both speed of computation and the number of DNA strands needed. Large-scale logic circuits can be constructed from the gates by simple cascading strategies. In particular, we have demonstrated a fast and compact logic circuit that computes the square-root function of four-bit input numbers.

Improving the Performance of DNA Strand Displacement Circuits by Shadow Cancellation.

DNA strand displacement circuits are powerful tools that can be rationally engineered to implement molecular computing tasks because they are programmable, cheap, robust, and predictable. A key feature of these circuits is the use of catalytic gates to amplify signal. Catalytic gates tend to leak; that is, they generate output signal even in the absence of intended input. Leaks are harmful to the performance and correct operation of DNA strand displacement circuits. Here, we present "shadow cancellation", a general-purpose technique to mitigate leak in catalytic DNA strand displacement circuits. Shadow cancellation involves constructing a parallel shadow circuit that mimics the primary circuit and has the same leak characteristics. It is situated in the same test tube as the primary circuit and produces "anti-background" DNA strands that cancel "background" DNA strands produced by leak. We demonstrate the feasibility and strength of the shadow leak cancellation approach through a challenging test case, a cross-catalytic feedback DNA amplifier circuit that leaks prodigiously. Shadow cancellation dramatically reduced the leak of this circuit and improved the signal-to-background difference by several fold. Unlike existing techniques, it makes no modifications to the underlying amplifier circuit and is agnostic to its leak mechanism. Shadow cancellation also showed good robustness to concentration errors in multiple scenarios. This work introduces a direction in leak reduction techniques for DNA strand displacement amplifier circuits and can potentially be extended to other molecular amplifiers.

Renewable Time-Responsive DNA Circuits.

DNA devices have been shown to be capable of evaluating Boolean logic. Several robust designs for DNA circuits have been demonstrated. Some prior DNA-based circuits are use-once circuits since the gate motifs of the DNA circuits get permanently destroyed as a side effect of the computation, and hence cannot respond correctly to subsequent changes in inputs. Other DNA-based circuits use a large reservoir of buffered gates to replace the working gates of the circuit and can be used to drive a finite number of computation cycles. In many applications of DNA circuits, the inputs are inherently asynchronous, and this necessitates that the DNA circuits be asynchronous: the output must always be correct regardless of differences in the arrival time of inputs. This paper demonstrates: 1) renewable DNA circuits, which can be manually reverted to their original state by addition of DNA strands, and 2) time-responsive DNA circuits, where if the inputs change over time, the DNA circuit can recompute the output correctly based on the new inputs, that are manually added after the system has been reset. The properties of renewable, asynchronous, and time-responsiveness appear to be central to molecular-scale systems; for example, self-regulation in cellular organisms.

Localized DNA Hybridization Chain Reactions on DNA Origami.

The field of DNA nanoscience has demonstrated many exquisite DNA nanostructures and intricate DNA nanodevices. However, the operation of each step of prior demonstrated DNA nanodevices requires the diffusion of DNA strands, and the speed of these devices is limited by diffusion kinetics. Here we demonstrate chains of localized DNA hybridization reactions on the surface of a self-assembled DNA origami rectangle. The localization design for our DNA nanodevices does not rely on the diffusion of DNA strands for each step, thus providing faster reaction kinetics. The locality also provides considerable increased scalability, since localized components of the devices can be reused in other locations. A variety of techniques, including atomic force microscopy, total internal reflection fluorescence, and ensemble fluorescence spectroscopy, are used to confirm the occurrence of localized DNA hybridization reactions on the surface of DNA origami. There are many potential biological applications for our localized DNA nanodevices, and the localization design is extensible to applications involving DNA nanodevices operating on other molecular surfaces, such as those of the cell.

Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

Design and Analysis of Localized DNA Hybridization Chain Reactions.

Theoretical models of localized DNA hybridization reactions on nanoscale substrates indicate potential benefits over conventional DNA hybridization reactions. Recently, a few approaches have been proposed to speed-up DNA hybridization reactions; however, experimental confirmation and quantification of the acceleration factor have been lacking. Here, a system to investigate localized DNA hybridization reactions on a nanoscale substrate is presented. The system consists of six metastable DNA hairpins that are tethered to a long DNA track. The localized DNA hybridization reaction of the proposed system is triggered by a DNA strand which initiates the subsequent self-assembly. Fluorescence kinetics indicates that the half-time completion of a localized DNA hybridization chain reaction is six times faster than the same reaction in the absence of the substrate. The proposed system provides one of the first known quantification of the speed-up of DNA hybridization reactions due to the locality effect.

Analog Computation by DNA Strand Displacement Circuits.

DNA circuits have been widely used to develop biological computing devices because of their high programmability and versatility. Here, we propose an architecture for the systematic construction of DNA circuits for analog computation based on DNA strand displacement. The elementary gates in our architecture include addition, subtraction, and multiplication gates. The input and output of these gates are analog, which means that they are directly represented by the concentrations of the input and output DNA strands, respectively, without requiring a threshold for converting to Boolean signals. We provide detailed domain designs and kinetic simulations of the gates to demonstrate their expected performance. On the basis of these gates, we describe how DNA circuits to compute polynomial functions of inputs can be built. Using Taylor Series and Newton Iteration methods, functions beyond the scope of polynomials can also be computed by DNA circuits built upon our architecture.