Design And Application Of Fluorescent Probes With Signal Amplification Strategy

Nucleic acid-based fluorescent probes have significant advantages including good water solubility,high thermal stability,ease of synthesis and modifications,with wide applications in biomedical research and biosensing.The single-stranded DNA/RNA fluorescent probes can not only hybridize with their complementary strands,but also function as the high-affinity recognition molecules to detect a variety of ions,small molecules,proteins and even live cells.Moreover,they can act as the substrates of enzymes and are used for enzyme identification,activity assay,mechanism study and inhibitor screening.In addition,they can induce signal amplification,transfering the biorecognition events?e.g.,hybridization,target binding and enzyme digestion?into the amplified fluorescence signals.In this thesis,we design a series of new nucleic acid-based fluorescent probes,and investigate how to reduce the background signal and interference as well as how to improve the detection specificity and sensitivity.We further apply these probes to detect a variety of disease-related biomarkers.?1?We developed a fluorescent method for simultaneous detection of multiple DNA glycosylases using two intrinsically fluorescent nucleotides labeled probes in combination with exonuclease-assisted recycling signal amplification.We designed a bifunctional DNA probe modified with one 8-oxo-7,8-dihydroguanine?8-oxoG?and five uracil bases,which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG.In addition,we designed the 2-aminopurine?2-AP?labeled signal probe and the P-dC labeled signal probe,which indicate the presence of hOGG1 and UDG,respectively.The presence of hOGG1and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals,with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG.This method can simultaneously detect multiple DNA glycosylases with the detection limits of 0.0035 U/mL for hOGG1 and 0.0025U/mL for UDG,and it can even measure DNA glycosylases at the single-cell level.Moreover,this method can be applied for the measurement of enzyme kinetic parameters and the screening of DNA glycosylase inhibitors,holding great potential for further applications in biomedical research and clinical diagnosis.?2?We developed a fluorescent approach with the integration of enzymatic labeling and single-molecule counting for identifying and quantifying a broad range of DNA damage types.We exploit the DNA glycosylases to remove the DNA damage and subsequently add the random biotinylated nucleotides?biotin-dNTPs?and fluorescent nucleotides?AF488-dUTPs?at the damage base site by the terminal deoxynucleotidyl transferase?TdT?in a template-independent manner,enabling accurate quantification of DNA damage level by single-molecule imaging.We overcome the limition of previous methods that cannot detect the clustered damage bases.This approach can detect the clustered damage bases no matter the positions of damage bases are farther or closer than one turn of helix.Moreover,this universal approach can detect various DNA damage types?e.g.,8-oxoG,uracil,and deoxyinosine?through variation of DNA glycosylase selected for recognition of a particular damage base and the same biotin-dNTPs label without requirement of appropriate dNTP for base-pairing with the opposite strand at the location of the damage bases.This approach shows excellent specificity and high sensitivity with a detection limit of 3.43×10^-16 M for the clustered damage 8-oxoG bases,and it can distinguish even 0.001%8-oxoG level from the complex DNA mixture.Importantly,this approach can be used to accurately ascertain the DNA damage level in different cancer cell lines and discriminate cancer cells from normal cells,holding great potential in the DNA damage research and the early diagnosis of DNA damage-related human diseases.?3?We developed a new fluorescent method for the detection of DNA methylation using one8-oxoG base-modified fluorescent probes on the basis of the enzeymes-assisted recycling signal amplification.The signal probe is modified with one 8-oxoG base at the opposite of the5-methylcytosine?5-mC?and labeled with ROX and BHQ2 at the 5?and 3?termini,respectively.The methylated cytosines remain unchanged after the bisulfite treatment,and they can subsequently hybridize with the signal probes to form the mC/8-oxoG base pairs.HOGG1can cyclicly cleave the 8-oxoG base in mC/8-oxoG base pairs to generate an enhanced fluorescence signal.In contrast,the unmethylated DNAs can be converted to uracils through bisulfite treatment,and subsequently hybridize with the signal probes to form the U/8-oxoG base pairs.The hOGG1 almost cannot cleave the 8-oxoG base in U/8-oxoG base pairs,thus,no significant fluorescence enhancement is observed in the presence of unmethylated DNAs.This approach exhibits good specificity and high sensitivity,and it can detect DNA methylation at single 5-mC resolution with a detection limit of as low as 3.458×10^-15 M.Moreover,this approach can distinguish as low as a 0.01%methylation level,and can detect DNA methylation in genomic DNA as well,holding great potential for accurate epigenetic evaluation and early cancer diagnosis.?4?We developed a universal fluorescent method for detection of lncRNAs and miRNAs with apurinic/apyrimidinic?AP?site modified fluorescent probes on the basis of enzymatic cascade amplification.The target ncRNA can be directly captured,easily separated and concentrated with the assistance of magnetic beads?MBs?,efficiently circumventing the contamination of genomic DNA.The capture DNA probe can be selectively cleaved into two or even more fragments by DSN,releasing the intact ncRNA and the cleaved ssDNA fragments with 3?-OH termini.The released ncRNA can hybridize with new capture probe to form the DNA/RNA heteroduplex,initiating another round cleavage of capture probes.Terminal deoxynucleotidyl transferase?TdT,a template-independent DNA polymerase?catalyzes the addition of dATPs at the 3?-OH termini of the cleaved single-stranded DNA fragments to obtain a long polyA sequences.The T-rich signal probe is modified with an AP site at the middle of the sequence and labeled with FAM and BHQ1 at the 5?and 3?termini,respectively.Each resultant long polyA sequence can hybridize with numerous T-rich signal probes to form a stable double-stranded DNA with an intact AP site,which can be cleaved by apurinic/apyrimidinic endonuclease?APE1?to release numerous FAM fluorophore.The cleaved signal probe with free 3?-OH termini can initiate a new TdT-mediated extension reaction and induce cyclic cleavage of signal probes to release abundant FAM fluorophores.This method shows high sensitivity with a detection limit of 0.072 fM for lncRNA and 0.06 fM for miRNA.It can be further used to quantify the endogenous miR-486-5p and lncRNA HOTAIR in cancer cells with a detection limit of 2 cells for endogenous lncRNA HOTAIR assay.