Optimizing Aptamer Sequence By Label-free Hybridization Probe Competition Method

Nucleic acid aptamers have been widely used due to their advantages such as high stability,in vitro screening,and a wide range of target molecules.However,the current problem is that it is difficult to obtain the core sequence of the aptamer,and there are lack of simple,fast and effective methods to optimize the nucleic acid sequence.Taking a nucleic acid aptamer that can recognize ochratoxin A(OTA)as an example,although many sensor and sensing methods have been developed based on this nucleic acid aptamer,there are too long and useless tail chains in the process of use to affect recognition.This work develops a label-free fluorescent aptamer sensing strategy for optimizing nucleic acid aptamer sequences.This strategy is based on the principle that SYBR Green Ⅰ fluorescent dyes are intercalated between complementary double strands to generate fluorescence.First,we put forward a set of evaluation criteria for complementary probes,because the signal changes caused by complementary short-strand probes and the affinity of the aptamers for their binding to OTA are not compatible.The competitiveness of short-chain probes is measured by(Amax-Amin)/IC50 value.Amax-Amin is the range of fluorescence intensity of the sensing method.The IC50 value represents the strength of the aptamer and the target molecule.(Amax-Amin)/IC50 value is greater,indicating that the complementary short-chain probe has a larger signal change range,and the aptamer shows good affinity for OTA under this short-chain probe.The optimal complementary probe is B13.Second,the core sequence obtained by tailoring and shortening the nucleic acid aptamer was reduced from the original 36 bases to 31.Third,research on the mechanism of aptamer recognition of OTA shows that the role of the G base at the 5 ’end of the aptamer is not a site to recognize OTA,but plays a key role in stabilizing the tertiary structure formed by aptamer recognition to OTA.Fourth,the results of previous studies and the results of this experiment predict that anti-parallel G-quadruplexes formed by OTA-induced nucleic acid aptamers,and formed three pairs of base-complemented blunt-end.Fifth,by extending the number of complementary base pairs at the tail end,a new nucleic acid aptamer with better affinity is obtained,which further validates our prediction of the tertiary structure of the nucleic acid aptamer.Sixth,using the different properties of OTA under acidic and alkaline conditions,the liquid-liquid extraction technology was improved,and the two-component liquid-liquid extraction technology of toluene and dichloromethane was used to pre-process the red wine sample.Seventh,using the optimized aptamer GG29CC to detect the actual red wine sample after sample preparation,the linear range of OTA is 1.25 to 15.63 nM,and the limit of detection is 1.25 nM.This sensing method has good selectivity for OTA and is not disturbed by OTB.In this paper,the OTA nucleic acid aptamer is optimized by a label-free hybridization probe competition method.This solution demonstrates a cost-effective high-throughput strategy that can obtain the core sequence of aptamers and further design new aptamers with high affinity.The new aptamer has a blunt-end,which will be more suitable for the design of new biosensors.Sensing method based on optimized nucleic acid aptamer has better limit of detection.