- James Lee Cho
Identification of Novel Peptide/RNA sequence targeting Malaria Parasite Proteins for Control of Malaria
Malaria is considered to be one of great threat of human health historically. Many trials of curing malaria disease with anti-malaria drugs have been executed however still not successful because of an ability of rapid malaria parasite resistance development by the genetic mutations or variations in their population. This is the reason why several novel peptide sequences that interrupt merozoite invasion into erythrocyte to cure malaria have been reported previously however it only lowered the merozoite invasion rate but it could not successfully stop the life cycle of malaria parasite for prevention or treatment of malaria, either. RNA-Peptide conjugation can be potentially a great way to increase the effect of inhibition rate of merozoite infection into erythrocyte and may even stop the life cycle of malaria parasite for annihilation of malaria disease in humans.
Malaria has been one of the serious parasitic diseases in human history1. 510 million people are suffering from this disease and 3 million people dies annually2. The protozoan parasite Plasmodium falciparum is the cause and Anopheline mosquito is the major vector for malaria3. Historically human immune system could not compete with the malaria parasites because human immune response is very complicated and differs based on endemicity level, genetic makeup and epidemiological factors4.
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It is not surprising that there have been many research about developing anti-malaria drug molecules for killing the parasites. However malaria parasites have been quickly become resistant to the drugs by mutating their genes. In the life cycle of malaria parasites in humans and mosquitos, there are many steps for parasites to develop and amplify. If even one step of the life cycle can be stopped, malaria parasite will fail to survive in their host organisms.
One of the steps in the life cycle has been targeted by related scientists is a merozoite invasion to erythrocyte step. Merozoite has a unique morphology for smoother invading to erythrocytes. Apical prominence part of merozoite plays a critical role of invading erythrocytes. Among many proteins in this region of merozoite, apical membrane antigen-1 (AMA-1) protein is highly considered to be involved in merozoite internalization into erythrocyte. This is the reason why AMA-1 has been a vaccine candidate protein for treating malaria for a long time. The exact mechanism of AMA-1 is not clearly understood yet however merozoite with antibody inhibited AMA-1 proteins showed a lack of ability for invasion into erythrocyte so it tells that AMA-1 does play a great role of merozoite invasion process.
In the previous research about targeting AMA-1 protein, through phage biopanning methods, several peptide sequences have been reported to successfully inhibit or interfere AMA-1 protein on the surface of merozoite, eventually inducing a lower rate of merozoite invasion into erythrocyte (About 60%). However it only lowered the infection rate but could not stop the life cycle. There has to be a better way to interrupt merozoite invasion for treatment of malaria disease.
Current Problems/Knowledge gaps
Considering the ability of malaria parasite to be quickly resistant to anti-malaria drugs, Current problem in malaria treatment is that it is really hard to find out an ultimate way to prevent or cure this disease. This is the reason why, as already mentioned in the introduction part, interruption of merozoite invasion has been tried in vitro by introducing novel selected peptide sequences, from phage bio-panning, to apical prominence of merozoite (AMA-1 region). However it could not successfully stop the invasion fully. However I believe that there could be better way to inhibit AMA-1 protein invasion process on merozoite.
Systematic Evolution of Ligands Exponential Enrichment (SELEX) is a great mean to seek a novel DNA or RNA sequence that binds to a target protein or cell. The main principle of SELEX is very similar to phage-biopanning. In SELSX, ramdomly synthesized DNA or RNA molecules are used for target instead of randomly displayed phage in biopanning. Lately, aptamer selection has been more advanced to be used for targeting more complex objects such as cells. RNA molecules can be successfully used to target merozoite cells.
RNA molecules can be used to target AMA1 protein on the surface of merozoite or merozoite cells themselves. Specificity of RNA molecules can be a lot higher than peptide molecules. That means RNA molecules can be better ones to interrupt AMA1 protein invasion mechanism successfully. It may estop the life cycle of malaria parasite, eventually induce the eradication of malaria parasite in human body and mosquitos. Even if it is true that there are still a lot of detailed information required for preventing and curing malaria5, my method can possibly improve the recovery rate of malaria patients in the world.
Gel mobility shift assay (EMSA) will be used to confirm the specificity of RNA molecules to AMA-1 protein. EMSA is one of great means to analyze interaction between RNA and protein. The principle of EMSA is that by observing the differences between each mobility of RNA molecules in polyacrylamide gel or agarose gel with and without a specific protein, Kd (Dissociation Constant) will be calculated. A high Kd value means that there is weak interaction between RNA molecules and the protein (Weak binding) and if Kd is low, it means that there is a strong interaction between RNA molecules and protein (Strong binding).
Community Requirement/ Hypothesis
If in vitro experiment of this research is successful, in vivo experiment with mice will be performed. After success of In vivo research, clinical trials will be performed.
Preparation of Merozoite6: The P. falciparum (3D7, D10, FAC-8, K1 and HB3) will be cultured through the method invented by Trager and Jensen7. Human serum supplement (Culture medium) will be prepared with 0.5% Albumax. Gas composition will be set to 1% O2, 5% CO2, and 94% N2. In the late stage of culturing parasite, parasite will be purified with Percoll cushion8.
Preparation of Phage Library6: M13 phage library will be purchased or obtained from a research group in University of Missouri at Columbia. M13 phage will be amplified by infecting XL_1 Blue E.coli. Phage will be shaking-incubated with tetracycline in LB (Luria Broth) medium. After incubation, only supernatant will be collected and used to precipitate the phage by mixing with Polyethylene glycol (PEG)/NaCl solution (33%). The phage can be re-suspended by distilled water to have a certain concentration.
Phage Biopanning9: 3 or 4 rounds of phage biopanning will be performed10. AMA1 protein (1.5 μg in 150μl coating buffer (0.1M NaHCO3, pH 8.5)) from the 3D7 strain of P. falciparum will be coated on the 96 well enzyme-linked immunosorbent assay (ELISA) plate for overnight incubation at 4oC. For blocking the well, blocking solution (0.5% bovine serum albumin (BSA) and 0.1M NaHCO3, pH 8.5) will be used before 3 times washings with PBS. Phage will be put onto the wells in 150μl probing solution (0.5% BSA in PBS) for 2hour slow shaking incubation at room temperature. In each round of biopanning, non-binding phage will be washed out with washing solution (PBS-T (0.5% Tween 20 in PBS). After 3 or 4 rounds of panning, Phage bound to AMA1 protein will be eluted with 150μl of elution solution (1.0M glycine HCl, pH 2.2) for 20 minutes at room temperature before neutralization with 10μl of 2M Tris. Eluted M13 phage will be amplified by infecting XL_1 Blue E.coli.
DNA sequencing and Analysis: DNA sequencing will be done by MCLAB Company. Qgene computer software program will be used to display the DNA sequence of genome of selected phage surface from biopanning. Through analyzing DNA sequences from the phage, dominant DNA sequence or pattern can be found and used to find out peptide sequence for their surface proteins. Once Novel peptide sequences or pattern are found out, they can be used for the further use through peptide synthesis.
Selected Peptide Synthesis: Peptide will be synthesized via Fmoc-based solid phase peptide synthesis (SPPS). In SPPS, Rink amide resin with HTCU coupling chemistry will be used in a scale of 0.25 mmol. Cleavage reagent (TFA, thioanisol, ethanedithiol, and anisol (95:5:3:2)) will be added for resin cleavage and immediate side chain deprotection. After filtration to separate resins, the resulting reaction mixture will be precipitated in cold ether to afford the crude peptide. The crude peptide will be employed in Semi-preparative scale reverse phase HPLC with solvent A (0.1% TFA in water) and solvent B (90%acetonitrile, 10%water, 0.1%TFA). The peptide will be purified by a linear gradient method. Electrospray ionization mass spectrometry (ESI-MS) and Analytical HPLC will be performed to confirm that peptide is pure enough to be used for the next experiment.
Systematic Evolution of Ligands Exponential Enrichment (SELEX)
RNA Selection from Library: RNA library can be simply purchased from a company or constructed in the lab. A synthetic DNA fool of various sequences can be purchased from QIAGEN Company. A T7 promoter sequence will be used to incorporate the 5’ primers in a reaction of the most conventional PCR cycle (30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C). After amplification, a T7 Transcription kit will be used and 2’-Fluoro (2’-F) pyrimidines will be incorporated for making more in vivo stable RNA molecules. The expected RNA aptamer size is about 80 bps.
In vitro RNA Selection and Amplification via PCR: 10nmol of RNA pool in 300 μl PBS will be added to the medium where merozoite cell cultured and blending-incubated for 30 minutes, following Hicke’s study. Bound RNA molecules will be eluted from the medium. The eluted RNA molecules will be added to the medium where erythrocytes are cultured. Only unbound RNA molecules will be collected. Reverse transcription, PCR amplification, and Re-transcription will be performed to generate novel RNA sequences (Molecules) that only bind to merozoite, but erythrocyte. Same steps can be repeated for RNA molecules to target pure merozoite AMA-1 protein.
Gel mobility shift assay (EMSA): Biotin labeled RNA molecules will be added to a buffer mixture solution (50mM Tris-HCl pH 7.0, 150mM NaCl, 0.25mg/ml bovine serum albumin). Each mixture solution with a different AMA-1 protein concentration will be incubated at approximately 37oC for 10 minutes before the addition of a dye mixture (Gel Loading Buffer II, Ambion) for electrophoresis at 70 voltage (at 25oC for 45 minutes) with a 1% agarose gel in 0.5 X TBE buffer (45mM Tris-boric acid and 1mM EDTA). RNA will be separated from the gel in the method, called standard capillary transferring. RNA molecules will be exposed to 302nm ultraviolet radiation on a UV transilluminator in about 3minutes to be cross-linked to the membrane (Zeta-probe nylon membrane from BIO-RAD). And then, the membrane will be submerged in the blocking solution at 30oC for 60 minutes (0.1 M Tris-HCL pH 7.5, 0.1 M NaCl, 2mM MgCl2 and 3% bovine serum albumin Fraction V)11. And the membrane will be mildly shaken in the blocking solution at 25oC for 12 minutes to be washed with 120ml of diluted AP 7.5 buffer with Streptavidin alkaline phosphastase (10μl) twice before washing with AP 9.5 buffer (0.1M Tris-HCl pH 9.5, 0.1M NaCl, 50mM MgCl2). The next incubation (15 minutes at 25oC) for the membrane will be completed with 8ml of AP 9.5 buffer, containing 2.5mg of nitroblue tetrazolium (NBT; Promega) and 1.25mg of 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Promega) before addition of TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0) to finish the reaction. The optical density of (color developed) biotin-labeled RNA band will be measured by ImageQuant, version 5.2 software11. The adjacent blank area need to be used as a control group. The optical density will be used to calculate for the molar concentrations of free RNA and RNA-AMA1 protein complex at equilibrium.
Novel RNA Binding Assay with Fluorescence Microscopy: Merozoite cells will be cultured on a cover glass in a plate for 2 days before 2 hour-incubation in serum-free medium. After removal of the medium, the cells will be washed three times with PBS. 4% paraformaldehyde will be used for cell fixation. AMA-1 protein on the cell surface will be dyed, using AMA-1 antibody bound to Fluorescin Isothiocyanate (FTIC) dye from Thermo Scientific (1:10 dilution). RNA molecules will be dyed with Toluidine Blue in 2x Dye buffer at 70oC for 10 minutes13. Cells with dyed PHB proteins will be incubated with dyed RNA molecules at 37oC overnight. Fluorescence images of the cells and RNA mixture will be taken with fluorescence microscope. Images of the other control receptor proteins (Tyrosine-Kinase Receptor and Erythropoietin Receptor protein on Erythrocyte) will be obtained after the same method is applied, using different antibodies in immunostanning method. Pure Protein can be used for conformation of specificity of RNA to AMA-1 protein in the same method.
RNA-Peptide conjugates for Inhibition of Merozoite invasion (In Vitro test):
I believe that my RNA-Peptide conjugates will successfully inhibit the invasion of merozoite into erythrocyte by interfering AMA-1 protein on apical prominence surface. Merozoite will not be able to thrive in human body because of this blockage of invasion mechanism of AMA-1 protein on the surface of merozoite.
Potential Problems and Solutions
Conjugation of RNA and Peptide via transthioesterification may not be very successful. Then other methods can be used for the conjugation. For instance, NHs ester-maleimide mediated conjugation or oxime formation through a hydroxyl-amine modified peptide reaction can be also used for linking RNA to Peptide.
Inhibition rate of RNA-Peptide conjugates may not be high enough to stop the merozoite invasion thoroughly. Higher concentration of RNA-Peptide conjugates can be used to possibly cause higher inhibition rate of merozoite invasion into erythrocyte by interruption of apical prominence related invasion mechanism.
6.Li, F.; Dluzewski, A.; Coley, A. M.; Thomas, A.; Tilley, L.; Anders, R. F.; Foley, M., Phage-displayed peptides bind to the malarial protein apical membrane antigen-1 and inhibit the merozoite invasion of host erythrocytes. J Biol Chem 2002, 277 (52), 50303-50310.
8.Dluzewski, A. R.; Ling, I. T.; Rangachari, K.; Bates, P. A.; Wilson, R. J. M., A Simple Method for Isolating Viable Mature Parasites of Plasmodium-Falciparum from Cultures. T Roy Soc Trop Med H 1984, 78 (5), 622-624.
9.Casey, J. L.; Coley, A. M.; Anders, R. F.; Murphy, V. J.; Humberstone, K. S.; Thomas, A. W.; Foley, M., Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes. Infect Immun 2004, 72 (2), 1126-1134.
Figure 1. Malaria Distribution in the World
Figure 2. The Life Cycle of Malaria Parasite
Figure 3. Cellular Structure of Merozoite
Figure 4. The Mechanism of Merozoite Infection to Erythrocyte
Figure 5. Overall Process of Phage Bio-panning
Figure 6. Overall Process of SELEX
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