Arinzechukwu Nwagbata's CIAP Aptamer Project (2017)

CIAP-APTAMER-CHLOROQUINE TARGETED DELIVERY FOR MALARIA PREVENTION

Introduction and Background

In 2015 only, there were 214 million cases of malaria around the world, resulting in an about 429,000 deaths. Malaria is a mosquito-borne infectious disease affecting humans and other animals caused by parasitic protozoans of the “Plasmodium” type. This disease is transmitted by infected female Anopheles Mosquitoes, and bites from these mosquitoes introduce the sporozoites (a stage in the life cycle of the plasmodium) from the mosquito’s saliva into a person’s blood. The parasites introduced into the bloodstream migrate to the liver where they infect hepatocytes (liver cells), mature, and reproduce. Once the organisms yield thousands of merozoites, they migrate from the liver into the blood undetected by sticking in the membrane of the liver cells. In the blood, they negatively affect the functions of the blood cells, resulting to symptoms that include fever, vomiting, headaches, yellow skin, seizures, coma, and/or death.

A 2006 in vitro and in vivo study conducted by the American Journal of Physiology showed that exogenous intestinal alkaline phosphatases quickly bound to the asialoglycoprotein receptors on hepatocytes (Tuin and Van der Vlag, 2006). Based on this discovery, the Calf-Intestinal Alkaline Phosphatase (CIAP) would bind quickly to the hepatocytes in the human liver, and with the aid of an aptamer of high affinity, can deliver drugs to break down sporozoites, prevent their maturation, or hydrolyze the bonds that attach them to liver cells, thereby preventing the reproduction and metastasis of these parasitic protozoans. If the CAIP-aptamer-drug complex is taken in regularly as a food supplement, this application could become the breakthrough to malaria prevention, and would cause a decrease in morbidity and maternal mortality caused by malaria.

The Calf Intestinal Alkaline Phosphatase (Figure 1) is an enzyme found in the intestinal walls of cows. It catalyzes the dephosphorylating of 5’ and 3’ ends of DNA and RNA phosphomonoesters. This enzyme is used in DNA sub-cloning, since DNA fragments that lack the 5' phosphate groups cannot ligate. CIAP has a molecular weight of 140 kda, and exists as a dimer. It is alkaline (most stable in buffers with basic pH), and thus, is a Zn2+ and Mg2+ dependent enzyme and can be used in the presence of Hepes buffer, PBS buffer, PCR buffer, etc. Its activity is enhanced in the presence of monovalent salts since it is negatively charged and interacts with nucleic acids which are also negatively charged. On the other hand, CIAP activity is decreased in the presence of reducing agents (DTT, β-ME), and metal chelators(e.g. EDTA), inorganic phosphate and phosphate analogs inhibit CIAP. There is an ongoing research for aptamer selection at the University of Texas at Austin. The aptamers selected with the CIAP target have different applications that range from therapeutics, diagnostics, and drug delivery. Eric Wei, Vincent Huynh, and Andrew Ellington, at the University of Texas at Austin, developed anti-CIAP aptamers that could be used to generate an inexpensive and robust CIAP-conjugated reagent for ELISA work and other molecular techniques that are AP-dependent.

Aptamers are small single stranded RNA or DNA oligonucleotides that can bind target molecules with high affinity and specificity. They can bind various target molecules, ranging from simple inorganic molecules to large protein complexes, and entire cells. Since aptamers are small with about 20 to 60 nucleotides, they are prone to degradation by various cytoplasmic components, and therefore, illnesses result due to insufficiency of these molecules. They also tend to bind other targets whose structures favor that of aptamers in the body of various organisms. This can as well, result to the malfunction of both the aptamers and the targets, thus, resulting in negative health consequences. Aware of these complications, scientists have engineered different methods of aptamer selection that allows for the development of these selected aptamers for different applications. The method of aptamer selection performed in this report is the Systematic Evolution of Ligands by Exponential Enrichment (SELEX, Figure 2). The sole purpose of this Bead-Based selection is to collect the most durable aptamers and amplify them. The selection process is conducted for about eight rounds, using the templates that resulted from previous rounds, thus, the repetition decreases variation. At the end of the final round, the resulting DNA molecules are sequenced, and the nucleotide arrangement of the most durable molecules are revealed.

The first stage of SELEX is Target Immobilization, and it involves the introduction of the protein target (CIAP) unto the beads, and the removal of unbound targets. This step is performed to select against targets with less binding affinity, since competition exists due to limited beads. The next step is Target and RNA pool incubation, during which RNA molecules from heat denatured RNA binding reaction, bind to the target. Another form of competition occurs here, but this time, between the RNA molecules; thus, we select against the molecules with less binding affinity. Afterwards, partition is carried out, and target- bound RNA is separated from unbound RNA during buffer washes that eliminate unbound species. The bound RNA is then collected (Elution) and Concentrated. Reverse Transcription is carried out, and single stranded DNA molecules are produced from the RNA molecules. These ssDNA molecules are Amplified with a Polymerase chain reaction (PCR) in order generate the ssDNA complementary strands, and to increase the concentration of the selected DNA molecules. Afterwards, a Transcription reaction is carried out, and mRNA is synthesized from these DNA templates. A new round of selection will then be conducted with the mRNA collected. Once the adequate number of rounds has been completed, the final dsDNA from amplification is sequenced. These stages are visually explained in fig.2.

So far, I completed the first round of selection, but will be repeating the PAGE to get more RNA products to move on to my second round. More rounds of selection will be conducted, and the selected aptamers with high binding affinity will be used to deliver Chloroquine to the hepatocytes where it will inhibit the life cycle of the plasmodium, and therefore, prevent malaria and its symptoms.

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References

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