High-efficiency and integrable DNA arithmetic and logic system based on strand displacement synthesis.
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ABSTRACT:
SUBMITTER: Su H
PROVIDER: S-EPMC6879481 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA logic gated operations empower the highly efficient analysis of multiplex nucleic acid inputs, which have attracted extensive attention. However, the integration of DNA logic gates with abundant computational functions and signal amplification for biomedical diagnosis is far from being fully achieved. Herein, we develop a bipedal DNA walker based amplified electrochemical method for miRNA detection, which is then used as the basic unit for the construction of various logic circuits, enabling the analysis of multiplex miRNAs. In the bipedal walking process, target triggered strand displacement polymerization is able to produce a large number of strands for the fabrication of three-way junction-structured bipedal walkers. The following catalytic hairpin assembly ensures the walking event and the immobilization of signal probes for output. Ultrahigh sensitivity is realized due to the integration of dual signal amplification. In addition, under logic function controls by input triggered cascade strand displacement reactions, NOT, AND, OR, NAND, NOR, XOR, and XNOR logic gates are successfully established. The as-developed DNA logic system can also be extended to multi-input modes, which holds great promise in the fields of DNA computing, multiplex analysis, and clinical diagnosis.
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Project description:DNA polymerase ? (Pol ?) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ? to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ? is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ? holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ? from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ? but not with Pol ?, RB69 gp43 or Pol ?. Neither was Pol ? able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ? is distributive. Our results suggest that Pol ? is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ? participates in short-patch base excision repair and ribonucleotide excision repair.
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Project description:Molecular circuits crafted from DNA molecules harness the inherent programmability and biocompatibility of DNA to intelligently steer molecular machines in the execution of microscopic tasks. In comparison to combinational circuits, DNA-based temporal circuits boast supplementary capabilities, allowing them to proficiently handle the omnipresent temporal information within biochemical systems and life sciences. However, the lack of temporal mechanisms and components proficient in comprehending and processing temporal information presents challenges in advancing DNA circuits that excel in complex tasks requiring temporal control and time perception. In this study, we engineered temporal logic circuits through the design and implementation of a dual cross-inhibition mechanism, which enables the acceptance and processing of temporal information, serving as a fundamental building block for constructing temporal circuits. By incorporating the dual cross-inhibition mechanism, the temporal logic gates are endowed with cascading capabilities, significantly enhancing the inhibitory effect compared to a cross-inhibitor. Furthermore, we have introduced the annihilation mechanism into the circuit to further augment the inhibition effect. As a result, the circuit demonstrates sensitive time response characteristics, leading to a fundamental improvement in circuit performance. This architecture provides a means to efficiently process temporal signals in DNA strand displacement circuits. We anticipate that our findings will contribute to the design of complex temporal logic circuits and the advancement of molecular programming.
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Project description:The strand displacement activity of DNA polymerase ? is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol ?-exo(-) to carry out strand displacement synthesis and discovered that it is regulated by the 5'-flaps in the DNA strand to be displaced. Under conditions where Pol ? carries out strand displacement synthesis, the presence of long 5'-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5'-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol ? with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol ? activities.
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