Isothermal Amplification Detection Of Protein Kinase And Long Noncoding RNA

The detection of biomarkers（e.g.,nucleic acids,proteins,enzymes,biological small molecules and even whole cells）plays an important role in early diagnosis,disease treatment,and prognosis.Generally,the content of these biomarkers in organisms is extremely low.Therefore,the development of accurate,effective,sensitive and simple detection methods is urgent.Isothermal amplification technologies achieve signal amplification by efficiently and rapidly accumulating nucleic acid sequences under constant temperature.Isothermal amplification-based fluorescent biosensors provide a multifunctional platform for the detection of biomarkers due to their advantages of good specificity,simplicity and speed,high sensitivity,and show great potential in promoting clinical diagnosis and drug development.In this thesis,we design two isothermal amplification-based fluorescent biosensors to achieve sensitive detection of cAMP-dependent protein kinase（PKA）and long noncoding RNA HOX gene antisense intergenic RNA（lncRNA HOTAIR）.The main contents of the two works are as follows:（1）We develop an ribonuclease HII（RNase HII）-driven single-ribonucleotide repairing-mediated cycling signal amplification strategy for sensitive detection of PKA activity in cancer cells.Protein kinase can catalyze the phosphorylation of peptides/proteins,and it is closely associated with various human diseases such as cancer,immune deficiencies,and Alzheimer’s disease.Sensitive monitoring of protein kinase activity is significant for biochemical research and drug discovery.In this assay,we design a peptide-DNA conjugate and a fluorescent probe labeled with Cy5 fluorophore and quencher 2（BHQ2）for PKA activity assay.In the presence of PKA,peptide-DNA conjugates can be phosphorylated,biotinylated phos-tag（a selective phosphate-binding agent）specifically recognizes and captures the phosphorylated peptide-DNA substrate,and the magnetic nanobeads（MBs）as the carriers can separate and enrich the phosphorylated peptide in complex matrices.The peptide-DNA conjugates as the catalytic substrates can translate the peptide signal to a nucleotide signal for the initiation of the RNase HII-driven cycling signal amplification.Signal probes are cyclically cleaved,resulting in the release of a large number of Cy5 fluorescent molecules.The combination ofMBs with the phos-tag-mediated phosphate group recognition can effectively eliminate the interference from the complex matrix,and the introduction of isothermal amplification technologies and single-molecule detection endows this assay with high sensitivity.This biosensor can achieve a detection limit of 1.98×10^-8 U/μL and a wide dynamic range from 1×10^-7 to 1×10^-2 U/μL.Moreover,this biosensor can be applied for the screening of PKA inhibitor and the measurement of cellular PKA activity,holding great potential in clinical diagnosis and biomedical research.（2）We develop a dual primer generation-mediated rolling circle amplification（PG-RCA）-triggered DNAzyme-induced the cleavage of signal probes for sensitive detection of lncRNA HOTAIR.HOTAIR is a non-coding RNA over 200 nt in length,which plays an important role in the occurrence and development of cancers and pathological processes（e.g.,cell differentiation,proliferation,and apoptosis）.It is highly expressed in a variety of human cancers（e.g.,prostate cancer,lung cancer,breast cancer）,and can be used as a clinical biomarker for early cancer diagnosis.Therefore,the sensitive detection of HOTAIR is of great significance.In this assay,when HOTAIR is present,the two padlock probes can specifically recognize the HOTAIR to form DNA/RNA double-stranded hybrid duplexes with a nick.This nick can be ligated by ligase,followed by the generation of two types of circular templates.Subsequently,the dual PG-RCA reaction can be triggered with assistance of polymerase and restriction enzyme,resulting in the production of abuntant DNAzymes.The resultant DNAzymes can cyclically cleave signal probes in the presence ofMg²⁺,releasing a large number of Cy3 molecules.The improved sensitivity of this fluorescent biosensor can be ascribed to the highly efficiency of dual PG-RCA reaction and the highly selectivity of DNAzymes-induced cleavage of fluorescent probes,and the detection limit is calcluated to be 0.678 fM.This assay can detect the expression level of HOTAIR in cancer cells,and can discriminate the tumor tissues of breast cancer patients and corresponding healthy adjacent tissues,which provides a promising platform for clinical diagnosis and biomedical research.