Functional Nucleic Acid-Based Nanoprobes For Study On Tumor Diagnosis And Therapeutic Efficiency Assessment

The development of rapid,specific and reliable tumor marker analysis methods is of great significance for the early cancer diagnosis and real-time monitoring of curative effects.Functional nucleic acids,as one class of recognition tools with special functions,have many advantages such as high biostability,convenient modification and easy design.They have shown unique advantages and amazing applications in biomedical research fields such as tumor diagnosis and targeted therapy.In addition,nanomaterials also have attracted much attention for bioimaging and tumor therapy due to their unique optical properties,high molecule loading efficiency,strong cell membrane penetration ability,and low cytotoxicity.Therefore,nanoprobes developed by combining functional nucleic acids with nanomaterials may have wide prospects in various applications in the field of tumor diagnosis and evaluation of treatment outcomes.To overcome drawbacks such as slow response speed,high false positive signal,poor specificity and poor stability,a series of novel nucleic acid-based nanoprobes were developed.These nanoprobes combine the advantages of functional nucleic acid and nanomaterials,localized acceleration effect and logic operation.As a result,rapid,high-sensitivity,and high-specificity tumor markers detection and imaging was relized in living cells,in vivo and in clinical samples.These methods provide a theoretical research basis for early accurate diagnosis of tumors as well as real-time evaluation of treatment efficiency.The detailed research contents are described as follows:1.Localized accelerated DNAzyme-based probe for amplified mi RNA detection in tumor cells.This study was carried out to develop a nanoprobe capable of overcoming the challenge of slow reaction speed and limited membrane-permeability caused by traditional nucleic acid-based probes in mi RNA detection.To address this challenge,DNA nanowire was combined with DNAzyme to develop a localized accelerated nucleic acid-based probe for rapid and sensitive intracellular mi RNA detection.In this design,both the DNAzyme and substrate strand are immobilized on the DNA nanowire by complementary DNA bases,which increases the local concentration of the probe,thereby speeding up the response.When the target exists,the mi RNA triggers the strand displacement reaction,activates the DNAzyme to cleave the substrate strand,resulting in fluorescence recovery.Subsequently,the released mi RNA binds with another substrate strand,triggering the next cycle of cleavage to realize a highly sensitive detection of mi RNA.The results showed that the sensitivity of this probe increased 1500 times compared with the non-amplified system,and the reaction time is shortened by six times.Furthermore,this nanoprobe can directly enter cells,thereby enabling imaging of mi RNAs in living cells with high sensitivity.2.Dual-mi RNA-driven DNA nanoprobe for precise differentiation of breast cancer subtype.Probes with the ability to detect only one target cannot discriminate between different subtypes of cancer cells.To solve this problem,a DNA nanoprobe with dual-mi RNA co-activation was constructed for the precise differentiation of different subtypes of breast cancer.The DNA nanoprobe consists of gold nanoparticles,a substrate strand,and a DNAzyme blocked by a target recognition sequence.In the absence of the target,the catalytic activity of DNAzymes is inhibited,and the gold nanoparticle quench the fluorescence of fluorophore.When mi R-21 and mi R-31coexist,they synergistically restore the catalytic activity of DNAzyme,thereby cyclically cleaving multiple substrate strands,resulting in the recovery of the fluorescent signal.Results show that this nanoprobe can quickly and accurately distinguish normal breast cells from high-metastatic and low-metastatic breast cancer cells.In addition,the nanoprobe was successfully used for rapid and accurate differentiation of different subtypes of breast cancer in living animals and clinical samples.This method is expected to provide a new molecular tool for rapid clinical screening of early breast cancer.3.DNA logic devices for specific imaging of endogenous metal ions and mi RNA in vivo tumor.The imbalance and abnormal distribution of intracellular metal ions can also cause a variety of diseases,so the specific imaging of metal ions in cells and in vivo is important for the diagnosis of diseases.Based on the aforementioned work,by combining a mi RNA-triggered entropy-driven cycling and Na~+-dependent DNAzyme-based amplification system,a DNA logic device for specific imaging of endogenous metal ions and mi RNA in tumor cells was developed.In the initial state,the fluorescence of the nanoprobe remains"OFF".When mi RNA and Na~+exist at the same time,a series of strand displacement reactions will be initiated,and the fluorophore will move away from the quencher,resulting in an amplified fluorescence signal.This method successfully realizes the simultaneous imaging of endogenous metal ions and mi RNAs in vivo tumor with high sensitivity,specificity and accuracy.This strategy may provide a new idea for analyzing low-abundant biomarkers.4.Aptamer-based granzyme B nanoprobe for real-time monitoring of cancer immunotherapy response.Functional nucleic acid-based probes can not only be used for the early diagnosis of diseases,but also for evaluating cancer therapeutic effects,which are able to provide theoretical guidance for timely changes in medicine usage.To overcome the poor correlation between the reported probe signal and immune activation response,a nanoprobe with targeted ability for real-time,non-invasive monitoring the tumors immunotherapy responses was developed by using an immune activation-related marker granzyme B as the target.In this design,aptamer and dye-labeled peptide are modified to the surface of gold nanoparticles through thiol-gold bond.When the granzyme B recognizes and cleaves the peptide sequence,the fluorophore will move away from the gold nanoparticle,and the fluorescence will be recovered.Intracellular and in vivo results showed that the signal intensity of the nanoprobe has a good linear relationship with the expression level of granzyme B and the number of cytotoxic T cells.This strategy is expected to provide a new method for the screening of clinical immune drugs.