Nucleases Participate In The Construction Of Synthetic Molecular Networks

Synthetic molecular network system is a key part of synthetic biology,which aims to build DNA molecular circuits.DNA molecular circuits is a precisely designed toolbox with good biocompatibility and programmability,and its powerful logic operation and signal amplification functions make it widely used in disease diagnosis and treatment.Currently,DNA molecular circuits operating at room temperature can only process single-stranded nucleic acid targets.However,the genomes of most biological samples are completely complementary double-stranded nucleic acid sequences,which cannot be used as input in DNA molecular circuits.This greatly reduces the substrate scope of DNA molecular circuits and limits its application fields.Molecular networks in living organisms require the participation of enzymes.Inspired by this,we designed high-performance DNA molecular circuits involving nucleases to respond to double-stranded nucleic acid input.We found that Lambda Exonuclease(λ exo)can drive the recognition of double-stranded nucleic acid input at room temperature,and by constructing the λ exo module,the single-stranded nucleic acid output which unrelated to the input sequence is replaced as a new input for subsequent DNA molecular circuits.We achieve its strict substrate preference by rationally optimizing the enzymatic reaction conditions of λ exo.When the double-stranded input is recognized by the λ exo module and forms the 5’-phosphorylated ds DNA substrate,it can be digested by λ exo to produce single-stranded output.The output,as a new circuit input,can generally respond to toehold-mediated strand-displacement(TMSD)logic operation circuits,hybrid chain reaction(HCR)amplification circuits and multi-enzyme involved circuits to achieve modular docking.In addition,we also found that the λ exo module can handle special double-stranded nucleic acids such as DNA-RNA complexes,showing a wide range of applications.The λ exo shows high catalytic efficiency of substrate conversion,which improves the operation speed of DNA molecular circuits,enabling it to realize molecular response and information processing in a short time at room temperature.All circuits obtain a fluorescent output signal within 15 minutes.In addition,the strong specificity of the nuclease effectively inhibits circuit leakage and can operate with almost none leakage.Compared with the existing DNA molecular circuits,the DNA molecular circuits constructed with the nuclease can quickly process double-stranded nucleic acid targets,and displace the single-stranded output to connect to a variety of widely used molecular circuits.This advantage not only expands the application range of DNA molecule circuits,but also own the properties of low leakage and simplified structure,thus have a better development prospect.