Research On Novel Detection Methodologies For Bioactive Molecules

In pace with the successful completion of the Human Genome Project (HGP) in 2000, proteomics raised the curtain on postgenomic era. The intense interests of proteomics are to find proteins with potential diagnostic and/or therapeutic significance, so as to foster a better understanding of disease processes, and to provide basis for early detection of disease, molecular typing, therapeutic efficacy and prognosis assessment. Besides, proteomics is also applied to discover new biological markers for drug action. To demonstrate the functions of extremely low expression of proteins during physiological and pathologic processes, there is an urgent need to establish methods for active proteins with high sensitivity and selectivity. The conventional assays are mainly antibodies-based on immunoassays. Nevertheless, as a new molecular recognition module, oligonucleotide aptamers are currently focused on and have shown their wide applications instead of antibodies in immunoassays.Aptamers are one type of single-stranded oligonucleotides (RNA or ssDNA) generated through an in vitro selection process termed SELEX (systematic evolution of ligands by exponential enrichment), and they can specifically bind to their target molecules, such as inorganic ions, organic small molecules, amino acids, polypeptides, proteins, and even whole cells. Nucleic acid aptamers have been named as"chemical antibodies"because they interact with their targets with high binding affinity and specificity comparable with the interaction between antibodies and antigens. Besides, aptamers have many unique advantages, such as ease of chemical synthesis and modification, superior reproducibility and stability, good tissue penetration and much low immunogenicity, and amplification capability. In this dissertation, based on aptamer 807-39 nt, which has been successfully in vitro isolated by our research group and can specifically recognize recombinant human erythropoietinα(rHuEPO-α), we describe a novel, universal and highly sensitive methodlogy for quantification of rHuEPO-α, which combined the structure characteristics with the amplification feature of aptamers.In addition, biotoxins are an important kind of poisonous substances produced by living organism. Besides being potential biological warfare agents for terrorist use, biotoxins could likewise be used as immunotoxins to treat cancer. Therefore, a rapid detection of biotoxins is an urgent need in military, counterterrorism and pharmaceutical fields, which is also a key research direction of our research group. Ribosome-inactivating protein (RIP) is a type of biotoxins that can inhibit protein synthesis and result in cell death. Taking ricin type II RIP for an example, we demonstrate an in vitro high sensitive assay for quantification of toxic RIP based on its N-glycosidase activity.This dissertation consists of four chapters.The first chapter is the introduction. In this chapter, we summarized the selection methods and features of aptamers and their applications in protein detection. The physical and chemical properties of ricin as a representative of RIP, mechanism of poisoning and different detection techniques were introduced as well. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.In chapter two, benefited from the specific recognition and the amplification capability of aptamers, we constructed two detection strategies for quantification of rHuEPO-αby magnetic beads-based aptameric real-time PCR assay. Strategy A is termed as"recognition-after-hybridization", in which a partial duplex hybridization of aptamer and biotinylated complementary sequence (CS) is immobilized onto Streptavidin MagneSphere? Paramagnetic Particles (SA-PMPs) via the biotin-streptavidin interaction. With the addition of rHuEPO-α, aptamer will switch its conformation to bind targets while the duplex will disassemble. As a result, aptamer will dissociate from PMPs and form a complex with target in aqueous solution. Upon magnetic separation, the dissociated aptamer can be collected for real-time PCR analysis. Strategy B is termed as"recognition-before-hybridization", in which aptamer is firstly incubated with rHuEPO-αto form a stable complex in an aqueous homogeneous solution. Biotinylated CS linked SA-PMPs is then used to hybridize the redundant unbound aptamer, acting as a"removing-the-background"module. After being separated with a magnet, a series of aptamers corresponding to various concentrations of rHuEPO-αare collected. The parameters including complementary sequences, the composition of binding buffer and ratio of CS and recognition aptamer sequence are optimized. After comparing two strategies on their sensitivity and feasibility, Strategy B was finally adopted as a preferred one to measure rHuEPO-α. The limit of detections (LODs) of 1 pmol/L and 6 pmol/L rHuEPO-α, and wide dynamic ranges from 6 pmol/L to 100 nmol/L and 30 pmol/L to 100 nmol/L were obtained for spiked binding buffer and matrix-half diluted artifical urine, respectively.If the target proteins exist in the real biological samples such as human serum, the high abundant matrix in biosamples would seriously block the amplification of real-time PCR. Otherwise, commonly used pretreatment techniques for nucleic acid samples are not suitable for above-mentioned strategy A or B. Following base-pairing principle, we designed and synthesized two complementary strands which could respectively bind primer regions at both ends of aptamer. The most suitable complementary strand was screened by electrophoretic mobility shift assay (EMSA), and linked to SA-PMPs via the biotin-streptavidin interaction, thus the formed probes could efficiently capture amplification templates from complex matrix. The results showed that this established pretreatment strategy could successfully help to determine the concentration of rHuEPO-αin human serum, in spite of a slightly lowered sensitivity.In chapter three, the universality of magnetic-based aptameric real-time PCR assay was validated. Based on high effeciency and specificity of binding characteristics between aptamer and target, aptamer were widely applied to various fields, e.g. analytical chemistry, but most aptameric methods were adopted to detect a single target molecule. There is still an urgent need to develop a universal aptameric assay. Based on the detection strategy described in chapter two, thrombin and ATP were chosen as detection targets for biomacromolecules and organic small molecules, respectively, to investigate the general applicability of such a strategy. The results demonstrated that previous described detection strategy could successfully determine the concentrations of thrombin and ATP spiked in binding buffer only after a simple optimization of hybridized sequences. Even for small organic molecule ATP, the size of that as same as a base, this strategy also has good feasibility.In the last chapter, a sensitive assay of bioactive RIPs was established on the basis of their N-glycosidase activity. RIPs are a kind of biotoxins that possess N-glycosidase activity and can specifically cleave nucleotide N-C glycosidic bonds at the general conserved residue in eukaryotic and prokaryotic rRNAs, blocking the synthesis of protein, and resulting in deactivation of ribosome. Since such proteins have extreme cytotoxicity and there is not yet any valid antitodes, it is of important realistic significance to establish sensitive and convenient assays for qualification, quantification and activity analysis of biotoxins. It should be mentioned that ricin (RCA60), type II RIP, is also the sole protein toxin listed in Schedule I of the Chemical Weapons Convention (CWC). A set of systemic qualitative and quantitative assays for ricin have been developed in our research group but lack of helpful tools to judge the activity (toxicity) of ricin. To overcome such a problem and further complete the systematic analysis of ricin, an in vitro analytical method of toxicity was established in virtue of its glycosidase activity, in which the toxicity of ricin was determined by measuring the release of adenine in depurination reaction by using LC-TOF/MS. Several parameters were systematically optimized, including conditions of LC and MS, internal standard, deoxyoligonucleotide substrate, pH, temperature, time, concentrations of protective protein and stop buffer of depurination reaction. Under the above optimized conditions, the active ricin at a concentration as low as 10 ng/mL was accurately determined, and the LOD could be reduced to 1 ng/mL with addition of immunocapture. We also found that ricin N-glycosidase in depurination reaction possessed high specificity, neither common proteases (including lysozyme, pronase and trypsin) or PNGase F andβ-Glucuronidase affected the stability of glycosidic bonds, and interfered with the depurination reaction of RIP toxins. Based on depurination reaction of ricin toxin in vitro, a rapid and sensitive analytical platform for toxicity analysis of RIP was developed. Several RIPs such as RCA60, ricin agglutinin (RCA120), ricin A chain (RTA) and abrin were tested in this platform. The results showed that this platform provided a convenient, rapid and sensitive assay for toxicity analysis of RIPs, and would meet the requirements for rapidly detecting activity of RIPs in the real biological samples.