Efficient Production Of Circular Functional Nucleic Acids And Its Application In Detection Of Pathogenic Bacteria

Pathogens caused millions of infections each year and greatly threaten human health.Therefore,it is very urgent to develop a simple,rapid and highly sensitive pathogenic bacteria detection technology.In the current detection methods,biosensing has attracted wide attention because of its advantages of miniaturization,low energy consumption and integration,etc.Circular functional nucleic acids(~CFNAs)have high biological stability,enhanced thermal stability,and the ability for replication by DNA polymerases in a rolling circle manner,which are widely used for biosensing.They are typically made from linear ss DNA molecules via ligase-mediated ligation.However,it is difficult to synthesize ~CFNAs with high selectivity and high yield because of complex tertiary structure of ~LFNAs and poor selectivity of ligase.Herein,we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a DNA aptamer that binds T4 DNA ligase,a DNA ligase widely used to perform DNA ligation reaction,and then by engineering this aptamer into a general-purpose templating DNA scaffold to“guide”the ligase to execute selective intramolecular circularization.We demonstrate the broad utility of this approach via the creation of a circular DNAzyme sensor for E.coli and a circular DNA aptamer reporter for toxin B(an established biomarker for diagnosing Clostridium difficile infections)with excellent detection sensitivity and specificity.In addition,a thermophilic fluorescence sensor based on the G4 DNA was constructed to enable the detection of single nucleotide polymorphs in pathogenic bacteria.The details are as follows:1.In vitro selection and characterization of DAS1.The 14th DNA pool was sequenced.The most dominant sequence DAS1 accounting for～24%,exhibited a high yield for the formation of its monomeric ring,~CDAS1.We also attribute the high circularization yield and selectivity to the fact that the SS1-J2/3 interactions are largely responsible.Futhermore,T4DL showed significantly better binding affinity to DAS1.Taken together,these results suggest that DAS1 has been successfully selected both as an optimal substrate for T4DL for circularization and an aptamer with a sequence-specified structure to bind T4DL.2.The synthesis of circular RNA-cleaving DNAzymes for E.coli biosensing.Circular EC1(named ~CEC1)was high-effectively cyclized by TDS1.T1.The biostability of ~CEC1 in CIM was investigated:As much as 90%of the ~LEC1 was digested in 1 hour,while only 13%of ~CEC1 was lost.And in the presence of E.coli,~CEC1-i was indeed able to cleave an RNA-containing sequence.It was able to detect E.coli at a concentration of 10~3cells/m L without cell culture.In addition,none of several arbitrarily chosen bacteria can activate ~CEC1 to cleave RDS,indicating high selectivity.Tests of clinical samples showed that our sensor holds great promise for clinical diagnosis.3.Engineered an effector-specific DNA switch MR-toxin B.MR-toxin B is made of separate Dapt1 and TDS1 domains that are conjoined by a weakened bridge element.MR-toxin B.4 carrying Bridge 4 produces a negligible circularization yield in the absence of toxin B,but exhibited a Y%of 45%on addition of ligand.The amount of ring products was increased with increasing concentrations of toxin B.In addition,no obvious ring products were observed when the switch was tested with unintended targets.We also examined the feasibility of performing quantitative analysis of toxin B using RCA.A limit of detection of0.1 n M was achieved within 30 min.4.We combined the thermal stable signal transduction unit with the isothermal amplifcation reaction to develop a thermophilic fuorescent assay.High temperature could ensure the accuracy of base pairing.Based on this,this fuorescent assay has been successfully applied for the identifcation of one-or two-mismatched base DNA.And it is expected to be generally applicable to identify SNPs in many other sequences,such as resistant gene.Furthermore,this work will open new opportunities for development of the thermally stable G4 DNA in biosensor.