Preliminary Research On DNA-scaffolded Linear Fluorescent Probes

It is a common phenomenon in cells that complex formed by multiple or diverse biomolecules performs specific functions, such as ribosome and spliceosome in cytoplasmic as well as the ion channels and receptor complex on membrane. Learning the mutual positional relationship and dynamic changes of their each part is critical for us to study their structures and functions. However, these complexes are typically nanoscale dimensions(usually less than 100 nm). At present, there are X crystal diffraction, electron microscopy and fluorescence resonance energy transfer technology that can complete the nanoscale detection, but they all have their own shortcomings. Laser scanning confocal microscope has become a conventional technology, but that can not be achieved due to the lack of resolution(over 250 nm).Our strategy is linear fluorescent probes of different lengths specific mark complexes of different molecules up, by linear position topography to enlarge the complex relationship between different molecules, so that lower-resolution fluorescence confocal microscopy can also be used to detect the location of the complex relationship between different molecules. As a preliminary and exploratory study, the main objective of this project is the synthesis of linear fluorescent probe and complete a preliminary application.We use different lengths dsDNA as a linear fluorescent probe. Its advantages including, firstly, synthesizing large amounts of DNA is convenient. Secondly, we can control the length of DNA accurately. Thirdly the diameter of dsDNA is only 2 nm.Fourthly there are many mature methods to make dsDNA fluorescence. Finally rich groups of DNA molecules can covalent bind to other small molecular substances, in order to achieve specific markers of target molecules. Thus, DNA molecules are well suited as a linear fluorescent probe material. In this paper, we use primers modified biotin groups and PCR instrument successfully synthesized a sufficient amount of three lengths linear probe(approximately 50 nm, 500 nm and 1200 nm respectively)and use two method(nucleic acid dye SYBR Green I and Rox) to make it become linear fluorescent probe. Gel electrophoresis assay proved three kinds of probes were synthesized successfully. Mass spectrometry result proved the 5 'end of the probe upstream primer connected biotin group successfully, and gel electrophoresis showed that these probes binding to streptavidin molecule specifically. Fluorescence intensityof the DNA probe increases with the DNA chain lengths and is far more than conventional dyes FITC by flow cytometry detection. Then, we used laser scanning confocal microscopy to observe DNA probes fluorescence effects on the silica beads which simulating cells, results show the fluorescence intensity increases with the DNA chain length. Further more, we observed DNA probes fluorescence effects on the erythrocytes and nuclear cells surface by confocal microscopy and found that effects of DNA probes is similar or stronger compared to the strong fluorescent reagent.In this study, we designed a new type of linear probe with the characteristics of DNA molecules. The probe aim at distinguishing the different components of a complex molecular structure by the different lengths of DNA probes. Not only simply using different fluorescence to distinguish, but also hoping to use this new linear probe to breakthrough limitations of existing fluorescence techniques. As a preliminary and exploratory study, we have synthesized the linear fluorescent probes successfully and detected the fluorescence effect. Follow-up study will choose suitable model of molecular complexes, two or more molecules of the complexes are labeled with the linear fluorescence probe specifically, direct imaging by laser scanning confocal microscope of to verify the linearity of different linear fluorescence probe.