Biosensors Based On G-triplex And Thioflavin T

The DNA or RNA nucleic acid sequence rich in Guanine(G)is an important class of biological macromolecules,which are abundantly present in the origin of genes(especially oncogenes),promoter regions,intragenic genes and telomere ends.It is widely involved in the regulation of gene transcription and translation,and is expected to become a new target for diseases,especially in cancer treatment and intervention.Therefore it has received extensive attention from the scientific community.A series of novel biosensing methods have been designed for the detection of microRNA and cocaine based on G-triplex and ThT.1)Chapter 2,G-triplexes have recently been identified as a new kind of DNA structures.They perhaps possess specific biological and chemical functions similar as identified G-quadruplex but can be formed by shorter G-rich sequences with only three G-tracts.However,until now,limited G-triplexes sequences have been reported,which might be due to the fact that their stability is one of the biggest concerns during their functional studies and application researches.Herein,we found a G-rich sequence(5'-TGGGTAGGGCGGG-3')which can form a stable G-triplex(Tm~60°C)at room temperature.The stable G-triplex can combine with thioflavin T and function as an efficient fluorescence light-up probe.Comparing with traditional G-quadruplex based probe,this triplex based probe was easy to be controlled and excited.Finally,the probe was successfully applied into constructing a label-free molecular beacon for miRNA detection.Taking advantage of these abilities of the G-triplex based fluorescent probe,the challenges faced during designing G-rich sequences based fluorescent biosensors can be efficiently solved.These findings provide important information for the future application of G-triplex.2)Chapter 3,High sequence homology among miRNA members challenges miRNA analysis.In this paper,we designed a G-triplex DNA structure based fluorescent probe,and constructed a label-free G-triplex molecular beacon(MBG3).The MBG3 with duplex-specific nuclease(DSN)was used to develop a reliable and highly selective amplification method for miRNA detection.Our MBG3 owned a short stem through excellent controllablity of G-triplex probe.The short stem protected MBG3 from DSN and efficiently minimising the problem of false positive signals without tedious modifications.Importantly,the improved recognition ability of MBG3,together with the sensitive substrate selectivity of DSN,make our novel method suitable for miRNAs detection.Wherein,the signal response of similar miRNA sequences with one-base difference has been reduced from 37% to 8% compared with traditional linear ssDNA probe-DSN-based method.3)Chapter 4,We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements.We have discovered that the cocaine-bindingaptamer can also bind the fluorescent molecule Thioflavin and thereby enhanced its fluorescence.Using this aptamer,we have developed a new sensor platform that relies on the cocaine mediated displacement of ThT from cocaine-binding aptamer as a result of competitive binding.We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 250 nM,which is 20 times lower than existing assays based on target-induced conformational change.Additionally,our assay is simple and cost-effective.