Construction Of Homogeneous Fluorescence Resonance Energy Transfer Ratiometric Nucleic Acid Probes Applied For The Detection Of Disease Markers

Homogeneous fluorescence resonance energy transfer（homo-FRET）occurs between two identical fluorophores.Theoretically,compared with the Heterogeneous fluorescence resonance energy transfer（hetero-FRET）,homo-FRET has been widely used in artificial light-harvesting systems to extend energy transfer distance and increase efficiency because the donor and acceptor have the same energy level without energy loss.Although homo-FRET has the ability to enhance FRET efficiency,it has rarely been used to construct corresponding nucleic acid probes for the analysis and detection of biological small molecules,proteins,and nucleic acids.DNA double helix structure,as a rigid and easily modified structure,can accurately control the distance of dye molecules.Therefore,it is commonly used to build various DNA structures for artificial light capture systems,such as DNA photonic wires,photonic networks and photonic circuits.Therefore,based on the characteristic of homo-FRET enhanced FRET efficiency,the following two ratiometric fluorescence sensing assays were carried out in this paper:1.A novel ratiometric fluorescent nucleic acid probe based on homo-FRET was designed for the detection of nuclease activity.Two Cy3 donors（D1 and D2）and one Cy5acceptor（A）were precisely modified on the double-stranded DNA template to construct D1-D2-A probes.According to the calculated F（?）ster distance（R₀）between Cy3 and Cy5,the distances of D1-D2 and D2-A were both designed as 5.0 nm.It was found that homo-FRET occurring between the two Cy3 donors effectively enhanced the hetero-FRET efficiency between Cy3 and Cy5（by approximately 10%）.Next,the constructed homo-FRET enhanced D1-D2-A probe was applied for the exonuclease Ⅲ（Exo Ⅲ）activity detection.Exo Ⅲ is an enzyme that has a high exodeoxyribonuclease activity for double-stranded DNA（ds DNA）in a 3’to 5’direction.The occurrence of homo-FRET between two Cy3 donors enabled the efficient energy transfer via hetero-FRET to the acceptor,corresponding with a high fluorescence intensity at 661 nm.When Exo Ⅲ was added,the D1-D2-A probe was hydrolyzed into single nucleotides,which disrupted the energy transfer process effectively producing a remarkable fluorescence change.The ratio of fluorescence intensity between Cy3 and Cy5（F_D/F_A）increases in an Exo Ⅲ concentration-dependent manner,which built the foundation of Exo Ⅲ quantification.The results showed that the target concentration showed a good linear relationship in the range of 0.25-8 U/m L,with the detection of limit was 0.17 U/m L（calculated according to the principle of three times the standard deviation,n=9）.Importantly,this platform also shows the potential for screening Exo Ⅲ inhibitors and detecting Exo Ⅲ activity in complex samples.The proposed homo-FRET enhanced ratiometric fluorescent probe has the advantages of simple operation,high selectivity,and high sensitivity.What’s more,this strategy for probe construction holds great promise for applications in DNA-related biochemical analysis and clinical research.2.A simple homo-FRET mediated three-donor-single-acceptor（3D-A）antenna nanostructure was constructed on the tetrahedral DNA framework（TDF）and applied to the quantitative measurement of hepatitis C virus（HCV）DNA via ratiometric fluorescence method.The three vertices of DNA tetrahedron were respectively modified with a Cy3 donor,and the recipient Cy5 was modified at the hairpin neck of the fourth vertex.Since the side lengths of TDF are all equal（4.4 nm）,strong FRET can occur among the four dyes.Moreover,homo-FRET among the three Cy3 donors can enhance the hetero-FRET efficiency between donors and acceptor,resulting in highe antenna effect.The target HCV DNA could open the modified Cy5 hairpin structure,increase the distance between the donor and acceptor,and cut off the FRET process.And the fluorescence emission intensity ratio（F_D/F_A）between Cy3 and Cy5 increased with the increase of HCV DNA concentration,which laid a foundation for the quantitative detection of HCV DNA.And the linear range of the 3D-A TDF antenna probe was 1–100nmol/L,with the detection of limit was 65 pmol/L（according to the principle of three times the standard deviation,n=9）.This method is a convenient operation,identifies single-base mismatches,and high sensitivity,which was comparable to the results of most HCR or CHA signal amplification methods.In addition,this method could also be used as a conventional method of DNA detection by changing the sequence bound to the target.In summary,this study constructed a ratiometric fluorescence resonance energy transfer nucleic acid probe based on homo-FRET for the sensitive detection of disease markers.The D1-D2-A double-stranded DNA structure proved that homo-FRET enhanced hetero-FRET efficiency,and the ratiometric fluorescence analysis method was established to determine the activity of exonuclease Ⅲ.By constructing a three-donor-single-acceptor（3D-A）antenna nanostructure on the DNA tetrahedron structure,the efficiency of homo-FRET and the enhancement to hetero-FRET was further improved.And the 3D-A TDF antenna probe was used for sensitive and highly selective quantitative analysis of hepatitis C virus DNA.In general,these methods broaden the scope of the application of homo-FRET based nucleic acid structures.