Screening And Identification Of Fibrin Aptamer—hirudin Carrier

Cardiovascular diseases including heart vascular and cerebral vascular diseases, are referred to as the circulation system function disorder, and charac terized by high incidence, high disability rate, high recurrence rate and high mortality. It is the primary cause of death worldwide, seriously threatening human life and health. In C hina, about 300 million people die each year from cardiovascular diseases, accounting for about 51% of the total deaths. Therefore, it is an important to develop drug to prevent and treat cardiovascular and cerebrovascular diseases.The risk factors of cardiovascular disease are quite large and complex, and thrombosis plays a key role in the development of this disease, so the effective prevention and treatment measures have a major significance to cardiovascular and cerebrovascular diseases. So far, the anticoagulants used in clinical for the prevention and treatment of thrombosis are two main categories: heparin(unfractionated heparin, low molecular weight heparin) and vitamin K antagonists(warfarin). But they are many defects, such as inconvenient administration, narrow therapeutic window, the slow onset, inducedthrombocytopenia and interactions with food drug easily. Therefore, it has become focus to develop a safe, effective and highly selective anticoagulants to prevent and treat thromboembolic diseases.The theory of meridian tropism has a long history, it can be traced back to “Yellow Emperor’s Canon of Medicine”, and it embodies the selective effects of drugs on the Zang Fu organs or a tissue of the body, which is the position of drug action. The effects of aptamer binding to the target, which is similar to those of the Traditional Chinese Medicine meridian tropism, can be considered as a new understanding of meridian tropism. This will promote the modernization of Traditional C hinese Medicine.Aptamers is a new medical molecular tools. It is screened from synthetic oligonucleotide library by using the systematic evolution of ligands by exponential enrichment(SELEX), with high specificity and high affinity. Aptamers can target to thrombosis and have a promising applications in the future. Hirudin extracted from the leeches, one of Chinese herbal drugs with activating blood circulation to dissipate blood stasis, is a potent and specific thrombin inhibitor with good prospect in clinical application. This study aimed to obtain the aptamers with specifical binding to fibrin through screening, used to carry Hirudin treatment of cardiovascular and cerebrovascular diseases.Objective:The cardiovascular diseases, caused by thrombosis have increased year by year. Inhibition of the thrombus formation has become a focus to prevent cardiovascular and cerebrovascular diseases worldwide. In this study, the fibrin in the coagulation pathway is targeted. Aptamers with high affinity and high specificity were screened from random oligonucleotide library using the systematic evolution of ligands by exponential enrichment(SELEX) technology. This may lay the foundation for developing thrombosis-targeted anticoagulant drugs.Methods:1. Nucleic acid library and primers were designed and synthesized. A single-stranded DNA(ss DNA) random oligonucleotides library of 99 nucleotides with 63 nucleotides random sequence at middle region and 18 nucleotides immobilized primer sequencesat both ends was constructed in vitro.2. Fibrin was coated by Dynabeads M-270 Epoxy, then the coating efficiency was determined by BCA method.3. The random oligonucleotides library was screened for aptamer using SELEX combining with the magnet. Then, the selection pressure had been strengthened(increased the t RN A, washing times, the frequency and shorten the reaction time of combining), repeated screening from last nucleic acid libraries and removed the non-specific binding of nucleic acid molecules with the fibrin in 6, 9, 12 cycle by adding blank beads. In the even cycle, the library was amplified with fluorescence labeling primers and the combination rate of nucleic acid library with the fibrin was calculated using indirect fluorescence quantitative method. The screening had not stopped until the rate did not increased any more.4. The aptamers screened from last cycle were amplified, purified, ligased with T vector and transformed into E.coli bacteria. Subsequently, single-clones were picked, incubated on plates with amp icillin for 12-16 hours and those aptamers had sequenced.5. The sequencs and features of the primary structure of the aptamers were analysed by Bio Edit software and the secondary structure by DNAMAN 8.0 software.6. Analyzsis of the specificity and affinity of the aptamers. The specificity of fibrin aptamers was indentified by dot immunogold filtration assay. Synthesized the aptamers labeled with biotin were combined with avidin-alkaline phosphatase conjugates. After chromogenic substrate enzymatic reaction, the absorbance at 450 nm was measured. According to the data, the conjunction curve and the dissociation constant Kd value were obtained by nonlinear regression analysis.Results:1. The single-stranded DN A oligonucleotide library of 99 nucleotide length with capacity of about 1017~18 was obtained.2. Fibrin was successfully coated by carboxyl magnetic beads with efficiency 87.65%.3. The fibrin aptamers with high affinity and high specificity were obtained after 15 cycles of pressure-increased screening.4. 57 clones of aptamers were obtained at the end of the fifteenth screening by cloning, transformation into E.coli, incubation for 12~16 hours at 37℃ and had sequenced by Invitrogen.5. The primary structure of aptamers aligned by Bio Edit software showed similar sequence, which may be the conservative sequences of aptamer and the structural basis for binding to fibrin.6. The secondary structure of the aptamers simulated with DNAMAN 8.0 software, showed that the main secondary structures were stem- loop, hairpin and G-quadruplex, but the stem- loop structure of most aptamers varied in size, location and the free energy.7. The specificity of aptamer identified with colloidal gold- labeled aptamers by dot filtration assay showed that the screened aptamers were highly specific. The affinity determined by biotin-avidin-alkaline phosphatase system with different concentrations of the target protein showed that fibrin aptamers were high affinity with the best dissociation constant Kd value was 43.12 nmol/L.Conclusions:A random single-stranded oligonucleotide library of 99 nucleotide length was designed and synthesized. After 15 cycles of screening, the fibrin aptamers were successfully obtained using SELEX with the magnetic beads as the medium by transformation and sequencing. The fibrin aptamers possessed high specificity and affinity with the target protein. This may lay good foundat ion for developing thrombosis-targeted drugs.