New Methods For Prion Protein Analysis Based On Ligands

Prion diseases, a group of fatal neurodegerative disorders, arised when cellular prion protein (PrP^C) undergoes some conformation rearrangement and formes the pathological isoform (PrP^Sc). The huge epizootic of "mad cow" in european and the discovery of variant CJD (vCJD, the transmission form of "mad cow" in human) at the end of last centrury, created enormours concerns among consumers and forced the European Commission to develop new simpler and faster diagnositc tests for prion diseases. Especially pre-symptomatic diagnostical that will do favors in the following aspects:firstly, the extent of epizootic would under surveillance by the goverment; secondly, ensure the safety of meat products, blood transfission, surgery, blood banks and plasma products; thirdly, identify prion diseases in the early stage so that the treatment could be initiated before the appearance of permanent brain damage. So simpler, faster and more accurate methods for prion diseases are in the ugent need of development. The application of conventional methods that are based on antibodies to pre-symptomatic diagnostical are limited by many reasons, such as the complicated procedure of antibodies producing, insufficient affinity and selectivity to targets. Aptamers, new-born nucleic acids ligands that bind to targets with high affinity and selectivity, are superior to antibodies in many aspects and thus has been widely applied to a variety of fields. In this contribution, aptamers are introduced to the analysis of prion diseases and a serial of prion protein detection methods have been developed based on aptamers. The main researches of this contribution include.1. Guanine-based aptamer beacon design and its application for PrP analysis. An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP^C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participatedhaprin structure consists of three segments.Namely, an aptamer sequence located in the loop, three guanine bases at 3'-terminal, and a fluophor modified at 5'-terminal. It was found that the guanine bases at the 3'-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5'-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrPC in the range from 1.1 to 44.7μg/mL with a limit of detection of 0.3μg/mL (3a).2. Label-free PrP detection based on aptamer and Hg²⁺. A new lable-free method for prion protein detection was developed based on the fact that T bases can interact with Hg²⁺ to form T-Hg²⁺-T structure. It was found that when aptamers were interact with Hg²⁺, intramolecular T-Hg²⁺-T structure was formed and thus the fluorescence of double-strand dyes such as Syber Greenâ… (SGâ… ) enhanced; however, the T-Hg²⁺-T structure destroyed with the addition of prion protein since the specifical interaction between aptamer and its target protein force aptamer underwent conformation change, and thus the fluorescence of SGâ… disappeared. The fluorescence intensities of SGâ… have good linear relationship with the concenctration of prion protein ranging from 13.0 to 156.0 nmol/L, R= 0.992.3. Compared with single-aptamer strategy, dual-aptamer strategy has higher affinity and selectivity and the employment of dual-aptamer strategy will dramatically increase the sensitivity and selectivity of aptamer-assays. Herein, dual-aptamer strategy was introduced to multi-targets analysis to solve the problem that limited the application of multi-targets analysis. Three targets, including ATP, thrombin and prion protien, are all related to diseases were selected for multi-targets analysis in this contribution. The results show that the detection of thrombin, ATP and PrP could be achieved simultanously in the same sample, and the analoges of the three targets such as adenosine, guanine, thymidine, IgG, snailase, BSA have on cross-reaction on the detection of targets, indicating the high selectivity of dual-aptamer strategy for multi-target analysis. 4. Dual-aptamer logic gate for the discrimination of prion-diseases-associated isoform. As the extension of logic gate, molecular logic gate that performs one or more inputs resulting from complex biological or chemical processes and produces a single output through logic operation, leaving the diagnosis of disease either "yes" or "no". Combining the advanteges of logic gate and dual-aptamer strategy, a dual-aptamer logic gate that is capable of OR and XOR logic operations (PrPc behaviors XOR logic operation while PrP^Res represents OR logic operation) has been designed. Compared with convential discrimination assays, the present dual-aptamer logic gate possesses the following advantages. Firstly, the dual-aptamer strategy could achieve highly selective discrimination of PrPRes in serum without isolation of target proteins prior to assay. Secondly, the dual-aptamer logic gate is simple and could be applied for prion diseases-associated isoform discrimination. Thirdly, the inputs (PrP and Gdn-HCl) and gates (MMPs-Apt1 and QDs-Apt2) used here are all cost-saving and chemical stable molecules, which are crucial for the development of smaller, more effective molecular-scaled computers. Fourthly, the gate (MMPs-Apt1) could be reused without loss of sensitivity since it only includes a surface-tethered monolayer of Apt1 without preferred conformation.5. Spectra characterization of the conformational changes of human cellular prion protein induced by heparin. Glycosaminoglycans(GAGs) is a ligand that distributes both in cell membrane surface and in endosomes. Up to now, the role of GAGs played in prions is still in controversy. By using heparin as an example, we investigated the interaction of GAGs and recombinant human cellular prion protein (rhPrP～C_23-231) by measuring the spectral features of the resonance light scattering (RLS), fluorescence and circular dichroism (CD). It was found that the intensity of both RLS and fluorescence get increased when the interaction of heparin with rhPrP^C_23-231 occurred and RLS intensities have good relationship with the concentration of rhPrP^C_23-231 ranging from 0.41 to 16.46μg/mL, with R=0.999. Meanwhile, the interaction between rhPrP^C_23-231and heparin make the fluorescence lifetime of rhPrP^C_23-231 extend, and the CD spectra indicate that heparin could induce the conformation rich in alfa-helix change to conformation rich in beta-sheet. In summary, we have developed a serial of prion protein detection methods based on aptamers. All of these methods are simple, fast, cost-saving and sensitivity, especially the dual-aptamer logic gate which are ultra-sensitive and selective for prion protein discrimination might further be applied to prion diseases pre-symptomatic diagnostical. Also, the researches present here extend the application of aptamers in diseases diagnosis.