Catalytic Hairpin Assembly-based Double-end DNAzyme Cascade-feedback Amplification For Sensitive Fluorescence Detection Of HIV-1 DNA

Objective:Human immunodeficiency virus type 1（HIV-1）,as the main pathogen subtype of acquired immunodeficiency syndrome（AIDS）,is highly infectious and strongly pathogenic.The accurate HIV-1 detection plays a vital role in diagnosis,treatment,and prevention of AIDS.At present,serological detection of specific immune antibodies produced by the body is the main method for laboratory diagnosis of HIV-1 infection.However,the"window period"before antibodies production hinders the early judgment of HIV-1 infection results.In addition,real-time quantitative PCR for HIV-1 nucleic acid does not miss detection during the"window phase",but it requires high personnel and equipment.To better meet the laboratory’s needs for HIV-1 detection methods,a simple and sensitive all-nucleic acid cascade-feedback amplification strategy for homogeneous and protein enzyme-free detection of HIV-1 related DNA（HIV-1 DNA）is proposed,which provides a new detection idea for early HIV-1 infection.Methods:1.Reaction process:In the presence of target DNA,through target-driven catalytic hairpin assembly（CHA）cycle reaction,the hairpins H₁ and H₂ are sequentially turned on to form H₁-H₂ hybrids with the active double-end DNAzyme,which further circularly cleave the ribonucleotide-containing quenched fluorogenic hairpin substrates to generate distinctly amplified fluorescence signal.Meanwhile,the released quencher-labeled fragments as target DNA analogues are also able to feed back and catalyze CHA-DNAzyme reaction process.2.Verification of amplification efficiency:By changing the part of DNAzyme sequences in H₁ and H₂ to poly-T sequences,the amplification efficiency of the generated single-end DNAzyme duplexes H_1T-H₂,H₁-H_2TT and double-end DNAzyme duplexes H₁-H₂ in the reaction system is compared;by replacing the part of the target sequence in H₃ to poly-T sequence,the effect of feedback amplification involving target DNA analogue on detection sensitivity is further verified.3.Evaluation of detection performance:After optimizing related experimental conditions such as H₃ concentration,temperature,time,etc.,the detection performance of this method,for example sensitivity,linear range,specificity etc.,are evaluated by detecting the fluorescence signal changes of target DNA with different concentrations.Results:1.The fluorescence intensity increment of double-end DNAzyme was over 3 times than that of single-end DNAzyme;the autocatalytic feedback amplification induced by target analogues could improve the sensitivity of CHA-DNAzyme sensing system.2.Under the optimal experimental conditions,the sensing method displayed a good linear range for target DNA from 1 pM to 2 nM with a detectable minimum concentration of 1 pM,and it also had high specificity as well as good detection repeatability.3.The recovery values of added HIV-1 DNA ranged from 94.48%to103.26%with relative standard deviations（RSD）between 2.02%and5.40%,indicating that the strategy held the potential for practical and reliable detection of HIV-1 DNA in complex biological matrices.Conclusion:In this work,an isothermal fluorescence sensing method was successfully constructed to detect HIV-1 DNA by combining CHA circuit,Mg²⁺-dependent DNAzyme cleavage reaction and the feedback effect of target DNA analogues.Due to the synergistic effect of multiple amplification designs,this method realized sensitive detection of target DNA.Considering the appealing feature of programmable nucleic acids,the current strategy may provide a prospective design for detection of broad-spectrum nucleic acid biomarkers.