Construction Of Molecular Beacons For Targeted Detection Based On Rolling Circle Amplification Of Aptamer Probes

Rolling circle amplification technology is developed in recent years by using circular DNA as a template to generate single-stranded DNA with many repeat sequences under the action of primers,four kinds of triphosphate nucleic acid and DNA polymerase.Function to improve detection sensitivity.A nucleic acid aptamer is a small molecule DNA or RNA that binds to a specific target,has high affinity and high specificity,and is widely used for analysis and detection of proteins,early diagnosis of tumors,and targeted administration.Molecular beacon(MB)is a new type of fluorescent probe developed by Tyagi and Krammer in 1996.It consists of two parts,the stem ring and the stem.One end modifies the fluorophore and the other end modifies the quenching group.When the stem portion is complementary paired,the fluorescence resonance energy transfer occurs when the distance between the fluorescent group and the quenching group is less than 10 n M,and fluorescence quenching occurs;when there is a complementary strand complementary to the stem ring,the hairpin structure is opened,Fluorescent quenching.Because of its simple operation,low noise and high sensitivity,it is used for the detection of RNA,amino acids,proteins and tumor markers,and has broad application prospects.Based on the rolling circle amplification technology,this paper modifies the aptamer sequence to the molecular beacon and constructs a molecular beacon for targeted detection.The main contents include the following aspects:(1)First,prepare a circular DNA template necessary for rolling circle amplification,perform loop-forming verification and template purification by polyacrylamide gel electrophoresis;use purified circular template,primer,d NTP,DNA polymerase and polymerase buffer Under the action,the rolling circle amplifies long single-stranded DNA with hundreds of repeats,characterizes the molecular weight of the product by agarose gel method,and characterizes the concentration and purity by a micro-ultraviolet spectrophotometer(Nanodrop).The morphology and flexibility of the product were characterized by transmission electron microscopy.(2)Thrombin is a specific serine protease that plays an important role in catalyzing many coagulation-related reactions.This part designed a 68-base molecular beacon with 15 thrombin aptamers with targeted detection of thrombin,mainly exploring fixed short-chain concentration,complementary strands of different lengths and different concentrations of molecular beacons.The degree of opening of the molecular beacon is the influence of the fluorescent signal,and its fluorescence intensity,signal-to-noise ratio and absolute value are characterized by a fluorescence spectrophotometer.The effect of different thrombin on the masking rate of fluorescence intensity was explored.The relationship between the masking rate and the thrombin concentration was obtained by fluorescence spectrophotometer.The detection limit was 0.078μM,which showed the rolling ring amplification technology.The advantage of molecular beacons in detecting sensitivity.(3)A novel molecular beacon with 83 bases was designed.This molecular beacon modified the AS1411 aptamer that can target cancer cells.The novel molecular beacon combines the signal amplification characteristics of the rolling circle amplification technology,the fluorescent "switch" of the molecular beacon,and the targeted recognition of the nucleic acid aptamer,which significantly improves the target compared with the general molecular beacon.Sensitivity,with accurate targeting and recognition capabilities,enables a sharp diagnosis of early tumor symptoms.This part mainly explores the dilution ratio of buffer,the fixed short-chain concentration,the complementary strands of different lengths and the influence of different concentrations of molecular beacons on the opening degree of molecular beacons,ie,the fluorescence signal,and its fluorescence intensity and signal noise by fluorescence spectrophotometer.The ratio is compared with the absolute value,and its morphology is characterized by transmission electron microscopy.(4)Based on the previous novel molecular beacons,the other end of the molecular beacon is modified with gold nanoparticles by a thiol group.Firstly,6nm gold particles were prepared by seed circulation method,and then the molecular beacon of 5-terminal modified FAM modified by thiol and 6nm gold were used to form MB/Au NPs complex by salt aging induction,and different salt concentration pairs were explored.The composite was characterized by UV spectrophotometer,Zeta potential meter and dynamic light scattering.The rolling ring amplification product was used to open the successfully bound MB/Au NPs complex to release the fluorescent signal.The fluorescence intensity,signal-to-noise ratio and absolute value were characterized by fluorescence spectrophotometer,and its morphology was characterized by transmission electron microscopy.