Quantitative Biology Study For Prion Protein Based On Prion-Aptamer Interaction

During the last decade, with the rapid development of life sciences, and the integration of which with a variety of subjects such as mathematics, physics, chemistry, etc., a new discipline-termed "Quantitative Biology" generated, in which scientists gradually focused on the a precise, quantitative study, seeking the universal law of biological systems. And the development of quantitative biology is of vital importance on the rapid development of the life sciences.Transmissible spongiform encephalopathies (TSE), or prion diseases, is a group of infectious neurodegenerative disorders accompanied by cognitive impairments, extensive brain damage and neuronal dysfunction. Although these diseases are rare, their unique mechanism of transmission and the concerns generated by the recent appearance of a new variant form of CJD, which has been linked to consumption of meat contaminated with BSE, have put prions in the spotlight. Increasing evidence now supports the idea that the central event is the conformational change of PrP through a posttranslational process, from cellular form (PrPc) to disease-causing conformation (PrPSc). Prion diseases always have a long incubation period before an extremely rapid clinical stage, which makes erlay diagnosis of the diseases is far to achieve. Simple, fast, and sesitive detection strategy of the diseases related protein could be helpful for rational design of novel therapeutic and diagnostic methods.In this contribution, seval novel prion protein detection strategys are proposed based on prion-aptamer interaction, which is simple, fast, sensitive. Additionally, we designed a axyzβ type peptides, which show great potential for the stability of a-helix. The mainly issues of this contribution are as follows: 1. New methods for prion protein detection based on PrP-aptamer interactionSurface-Enhanced Raman Scattering Investigations on Prion Protein-dual Aptamer Interaction Surface-enhanced Raman scattering (SERS) spectra, which can provide large information on the chemical structure of the probed substances, have been successfully performed as a well-established tool in complex biological system. PrP is reported possessing two different distinct binding epitopes for two aptamers. One aptamer (Aptl) recognizes epitope23-90of the N-termina. while the other one (Apt2) specifically binds with the90-231of prion, which is corresponded to the β-sheet structure of PrP. Considering the strong Raman activity, Coomassie brilliant blue (R-250) was employed to label target proteins and probe protein-aptmer interactions, and an original quantitative study to investigate the interaction between PrPc and its two aptamers was develped based on SERS spectroscopy. Experimently, Aptl and Apt2are conjugated to silica coated silver (Ag@Si) NPs, followed by the addition of R-250labeled PrP. After well mixed with the target proteins, the aptamers undergo a conformational transition from an unfolded structure to a folded conformation, which possesses high affinity with the target, leading to the nanoparticles aggregation, sequentially inducing enhanced raman scattering. This strategy, which is addressing two aptamers recognizing distinct epitopes of the target, is simple, rapid, and high specificity to probe protein-aptamer recognition in the solution.Metal-enhanced fluorescence of nano-core-shell structure used for sensitive detection of prion protein with a dual-aptamer strategy. We developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the prescence of prion protein, core-shell Ag@SiO2. which are functionalized covalently by single stranded aptamer (Aptl) of prions and Cyanine3(Cy3) decorated the other apater (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of particles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission retes of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of50-300μM, which is successfully applied to the detection of PrP in the mice brain homegenates. as well as in the live cells. Ultra-sensitive detection of prion protein with a long range resonance energy transfer strategy. We developed an aptamer-mediated Long range Resonance Energy Transfer (LrRET) strategy to achieve the detection of cellular prion protein (PrPc) at attomolar level, which could be extended to the detection of other analogous proteins. Under optimal conditions,0.82-3.30fM of PrPccan be quantitatively detected with the limit of determination of33aM (3a). The strong fluorescence signals of QDs could be distinguished byconfocal imaging in vivo, and efficiently quenched with the addition of AuNPs-Apt conjugate. The visual LrRET system is promising to real-time trace the endogenous cellular prion protein.2. Interaction studies of the peptides and prion proteinUntil recently, no effective treatment strategy is avaliable for prion diseases, while the widely accepted therapautic method is. based on the stability of a-helical conformation, and the inhibition of transformation between a-helical and β-sheet Herein, we designed four axyzβ type peptides for sequence range of182-190of1801prion protein (a valine to isoleucine mutation), which plays an important role in the pathological prion disease, xyz is composed of hydrophobic amino acids (aa) that are found in the prion protein, while a/β stands for basic or acidic amino acids, which can interact with the relevant acidic or basic aa in prion protein to form salt briges. And the existence of salt briges lead to the shortening of protein length, and make it more stable.The results showed that only the sequence of EVVKK can bring a very good stability, and induce the conformation changes from β-sheet to a-helix, while the effect of other three peptides can be neglected, which can act as a reference for the development of peptide drugs, and further for the clinical treatment.