Construction Of Nucleic Acid Aptamer Libraries And Preliminary Screening Of Single Strand DNA Sptamer For Zearalenone

Zearalenone(ZEN)is a mycotoxin produced by Fusarium,could be found in foods and feedstuffs worldwildely,exspecially in corn,wheat,barley,millet,oats and their releated products.As a toxin zearalenone has many side-effects,which could cause serious problem for human health,such as reproductive toxicity,immunotoxicity,hepatotoxicity,nephrotoxicity,cytotoxicity,and also could induce to the disorder of endocrine system and resulted in tumor.Therefore,it is of great meaningful to develop rapid,specific,sensitive and quantification detection methods for the on-site determination of ZEN.As a new type of molecular recognition element,aptamers have many characterism advantages over antibodies: such as variable types of target molecules,high affinity and specificity,easy production,good stability and much capable for modification.Aptamer is an oligonucleotide sequence that has been screened in vitro and is capable of binding with a corresponding target with high affinity and strong specificity.The aptamer can be either DNA or RNA and typically 15-100 nucleotides long,which can form specific,complex three-dimensional structures.Based on the three-dimensional functional structure,it can combine with a variety of target molecules to form a stable complex.Combining with the development of biosensor technology,aptamers have been employed in a variety of novel detection methods development,which have been widely used in the fields of medicine and food safety.In this study,zearalenone aptamers were screened using graphene oxide-SELEX and affinity column-SELEX.In the screening program for graphene oxide-SELEX,the ss DNA library containing 44 random nucleotide sequences with a total length of 80 nucleotides was chemically synthesized in vitro.Graphene oxide was used as the screening medium and ZEN was the target molecule.A total of 19 rounds of screening were performed.The screening products of the sixth and nineteenth rounds were cloned and sequenced.The 42 positive clones of the sixth round were sent for sequencing to obtain 40 sequences and 31 different sequences;the nineteenth round 46 positive clones were sent for sequencing to obtain 45 sequences and 27 different sequences.A partial sequence was selected for activity identification using the SYBR Green I method and the results showed that none of the selected sequences were bound to ZEN.For the method of affinity column-SELEX screening scheme,a three-segment library with a length of 91 nucleotides is used.In the middle of the library is a segment of a 19-nucleotide fixed sequence.The left and right sides of the fixed sequence are ten and twenty nucleotide random sequence,respectively.And the ends of the library are fixed primer binding sequences.The screening method is to fix the screening library by complementary sequences,and then let the target molecules compete with the complementary sequences to elute the sequences of the binding target molecules.During this screening process,both agarose gel electrophoresis and fluorescence were used to monitor whether the library was successfully immobilized.A total of 17 rounds screening have been conducted,on the 16 th round of screening,the efficiency reached to 40 %.The bioactivity of the selected ssDNA aptamer were identified by the SYBR Green I assay and the structure-switching fluorescence assay.Unforturnatly,none of the selected aptamer showed binding capability for the ZEN.