Surface Modified Magnetic Particles In The Studies Of Small Molecule Interactomes

We described in this paper the application research of using magnetic iron oxide nanoparticles conjugated with the natural product, effective component of Chinese traditional medicine, Huperzine-A(Hup A) and dinucleoside tetraphosphate(Ap4A) as carriers,respectively screening the target of Hup A from phage cDNA library and brain tissue lysate of mammalian animal, and the target of Ap4A from prokaryotic cell lysate and eukaryotic tissue lysate through magnetic separation. The interaction between Hup A and the screened specific phages was analyzed through capillary electrophoresis(CE), and the interaction between Hup A and the screened protein expressed and purified in vitro was identified through surface plasmon resonance(SPR).The magnetic particles with carboxyl groups were prepared through the method of chemical coprecipitation using the polymer synthesized using lactic acids by photocatalyst as templates, and their surfaces can be modified by all kinds of bioactive molecules through the reaction between carboxyl group and other chemical groups. The modified particles were stable in solution and paramagnetic, so they have much potential in biological separation and medicine.Functional small molecules, especially natural products, are the main source of modern medicine. They played such an important role in helping human beings to overcome diseases even in the condition of threatening life. The functions of natural products are usually judged from their origins or are approved from associated disease models, but the exact molecular targets are unknown for most natural products, and their action mechanisms are also unclear, which forms a certain barrier for looking for or synthesizing the analogues, substitutes, or better ones of natural products, also for further revealing the causes of diseases. Therefore, establishing more effective target identification methods of natural products to find their possible unknown targets through combining with the existing techniques has become the key step of accelerating modern medicine process.In this paper, we first conjugated the effective component of Shezushishan, Hup A on the surface of magnetic nanoparticles, and screened its targets using magnetic separation and biopanning from phage cDNA library and brain tissue lysate of mammalian animal.2specific phages were repeatedly screened out after several rounds of magnetic biopanning. The proteins located by these2specific phages were found through PCR, gene sequencing and blasting in gene bank. CE was used to analyze the in vitro interaction between the2specific phages and any one unspecific phage and Hup A, and the result indicated a certain binding between2specific phages and Hup A, and nearly no binding between the unspecific phage and Hup A, which further removed the possibility that specific phages were the result of non-specific absorbance of magnetic particles. The functional protein, mitochondrial NADH dehydrogenase subunit1, located by the gene displayed by one specific phage, was expressed and purified through prokaryote expression and its interaction with Hup A was also tested by SPR. A certain binding constant was got and indicated a certain interaction between this protein and Hup A. Another functional protein, mitochondria ATP synthase, was identified again from eukaryotic tissue lysate. All these results, combined with other research about Hup A'functions, to a large extent illustrated that Hup A could be used to cure AD or other neural degenerative diseases by adjusting mitochondria activity. Some functional mitochondria proteins, mitochondrial NADH dehydrogenase and ATP synthase, might become Hup A's new targets.Besides Hup A, we synthesized biotin-Ap4A in vitro, and conjugated it on the surface of magnetic particles with streptavidin. The targets of Ap4A were screened from prokaryotic cell lysate and eukaryotic tissue lysate through magnetic biopanning. One specific protein was screened out repeatedly after6times biopanning for prokaryotic screen, and4specific proteins were screened out for eukaryotic screen. After being identified by Mass spectrometry (MS), Ap4A's known target, GroEL appeared in the protein list, which not only indicated the feasibility of applying magnetic biopanning technique to screen small molecules's targets, but also increased the possibility of other identified proteins, especially IMP dehydrogenase and eukaryotic Hsp family proteins to become Ap4A's targets. Indeed, the experiment in this part is another trial of applying magnetic particles to identify targets, and it is also the extension and further verification of this method applied in Hup A's target identification.The experiments from these two parts fully indicated that the affinity biopanning chromatography using magnetic particles as affinity matrix, through combining with phage display and MS based proteomics technique, will become a new effective method of identifying the targets of bioactive molecules, especially of natural products.