Sequence Truncation And Application Research Of Aptamers

The application of long-length aptamers was usually limited,because the redundant nucleotide sequence lead to the degradation of aptamer performance.Appropriate truncation for long-sequence aptamer based on retaining the binding domain,could improve its affinity and specificity,and then extend application.This work truncated the original aptamers for 25-HydroxyvitaminD3 and 17?-estradiol(E2),and performed the detection for 25-HydroxyvitaminD3 and E2 by label-free fluorescent method and AuNPs-based colorimetric method,respectively.The results of this work will promote the application and industrialization of aptamers.(1)Vitamin D insufficiency is closely related to various kinds of metabolic diseases.Acted as a marker of vitamin D status,25-HydroxyvitaminD3 detection possesses important practical significance.In this work,highly sensitive fluorescent detection of 25-HydroxyvitaminD3 using truncated affinity-improved aptamers were developed,based on fluorescence intensity changes of PicoGreen(PG)generated upon binding to double-stranded DNA(dsDNA)formed through hybridization of aptamer and corresponding complementary strand.Four truncated aptamers were obtained by intercepting the small hairpin loop as the binding domain and retaining double helix structural domains of different lengths that exist in the selected original 25-HydroxyvitaminD3 aptamer.Under the optimized PG concentration,we conducted comparison experiments for affinity and specificity of these four truncated aptamers and original aptamer.Among them,the shortest aptamer with only 21 mer,D3-4,was found to show highest affinity and specificity to 25-HydroxyvitaminD3,with the limit of detection of 0.04?g/mL,which solved the problem that original long aptamer could not applied for this fluorescent detection of 25-HydroxyvitaminD3.The truncated 25-HydroxyvitaminD3-specific aptamer with highly enhanced affinity performs promising application in sensitive detection of 25-HydroxyvitaminD3.(2)E2 residues could enrich in organisms via food chain and lead to harmful biological effects to human body.To ascertain the binding domain of original E2 aptamer(E00)with long-sequence,we developed novel truncated E2 aptamers from EO0,through rationally designed truncation by intercepting a single ring or a combination of rings(containing hairpin loop,interior loop or multiloop)at different sites and retaining appropriate double helix regions.Through comparison,15 mer E09 presented improved affinity and higher specificity,indicating the hairpin loop near to 3'end of EO0 served on the specific binding domain to E2.E09 was used for AuNPs-based colorimetric determination of E2,achieved the detection limit of 0.02?g/mL.Along with simple pretreatment,E09-based aptasensor was applied for E2 detection in milk and mineral water.The proposed truncated aptamer has great application potential in E2 detection,and this work provides designed truncation reference for other long-sequence aptamers.