Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min−1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

[1]  Wong Cheng Lee,et al.  High-throughput cell cycle synchronization using inertial forces in spiral microchannels. , 2011, Lab on a chip.

[2]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[3]  A. Bhagat,et al.  Inertial microfluidics for continuous particle separation in spiral microchannels. , 2009, Lab on a chip.

[4]  Stephen J Rogerson,et al.  VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum. , 2006, Molecular and biochemical parasitology.

[5]  Han Wei Hou,et al.  Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation. , 2011, Lab on a chip.

[6]  Thomas S. Rask,et al.  Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children , 2012, Proceedings of the National Academy of Sciences.

[7]  M. Frank,et al.  Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production , 2006, PLoS pathogens.

[8]  Terence P Speed,et al.  Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites , 2006, Genome Biology.

[9]  Patrick E. Duffy,et al.  Adherence of Plasmodium falciparum to Chondroitin Sulfate A in the Human Placenta , 1996, Science.

[10]  L. Rivière,et al.  Antigenic variation in Plasmodium falciparum. , 2008, Annual review of microbiology.

[11]  A. Craig,et al.  Exported Proteins Required for Virulence and Rigidity of Plasmodium falciparum-Infected Human Erythrocytes , 2008, Cell.

[12]  Thor G. Theander,et al.  Evidence for the Involvement of VAR2CSA in Pregnancy-associated Malaria , 2004, The Journal of experimental medicine.

[13]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.

[14]  Michael Musheev,et al.  Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers. , 2005, Journal of the American Chemical Society.

[15]  John R Yates,et al.  Proteomics approach reveals novel proteins on the surface of malaria-infected erythrocytes. , 2004, Molecular and biochemical parasitology.

[16]  Travis Harrison,et al.  A Host-Targeting Signal in Virulence Proteins Reveals a Secretome in Malarial Infection , 2004, Science.

[17]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[18]  Joseph M. Martel,et al.  Inertial focusing dynamics in spiral microchannels. , 2012, Physics of fluids.

[19]  D. Di Carlo.  Inertial microfluidics. , 2009, Lab on a chip.

[20]  Melanie Rug,et al.  Targeting Malaria Virulence and Remodeling Proteins to the Host Erythrocyte , 2004, Science.

[21]  Gwendolyn M. Stovall,et al.  Technical and biological issues relevant to cell typing with aptamers. , 2009, Journal of proteome research.

[22]  N. Mohandas,et al.  Red blood cell glycophorins. , 1992, Blood.

[23]  Michael Lanzer,et al.  A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A , 2005, EMBO reports.

[24]  Joel H. Janes,et al.  A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells , 2012, Proceedings of the National Academy of Sciences.

[25]  B. Sullenger,et al.  Generation of species cross-reactive aptamers using "toggle" SELEX. , 2001, Molecular therapy : the journal of the American Society of Gene Therapy.

[26]  A. Cowman,et al.  The adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A is mediated by P. falciparum erythrocyte membrane protein 1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[27]  H. Ozaki,et al.  Sialyllactose-binding modified DNA aptamer bearing additional functionality by SELEX. , 2004, Bioorganic & medicinal chemistry.

[28]  A. Bhagat,et al.  Continuous particle separation in spiral microchannels using Dean flows and differential migration. , 2008, Lab on a chip.

[29]  R. Tompkins,et al.  Continuous inertial focusing, ordering, and separation of particles in microchannels , 2007, Proceedings of the National Academy of Sciences.

[30]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[31]  Steven W Graves,et al.  Inertial manipulation and transfer of microparticles across laminar fluid streams. , 2012, Small.

[32]  Gwo-Bin Lee,et al.  Integrated microfluidic system for rapid screening of CRP aptamers utilizing systematic evolution of ligands by exponential enrichment (SELEX). , 2010, Biosensors & bioelectronics.

[33]  Y. Zhan,et al.  Vortex-assisted DNA delivery. , 2010, Lab on a chip.

[34]  X. Su,et al.  Malaria biology and disease pathogenesis: insights for new treatments , 2013, Nature Medicine.

[35]  H. Amini,et al.  Inertial microfluidic physics. , 2014, Lab on a chip.

[36]  K. Haldar,et al.  Malaria: mechanisms of erythrocytic infection and pathological correlates of severe disease. , 2007, Annual review of pathology.

[37]  Kohei Ogawa,et al.  Numerical study on development of particle concentration profiles in a curved microchannel , 2006 .

[38]  R. Tompkins,et al.  Equilibrium separation and filtration of particles using differential inertial focusing. , 2008, Analytical chemistry.

[39]  Zbynek Bozdech,et al.  A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells , 2012, Proceedings of the National Academy of Sciences.

[40]  A. Heeger,et al.  Micromagnetic selection of aptamers in microfluidic channels , 2009, Proceedings of the National Academy of Sciences.

[41]  Hidesato Ito,et al.  Flow in curved pipes. , 1987 .

[42]  T. Gilberger,et al.  Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export , 2013, PLoS pathogens.

[43]  Han Wei Hou,et al.  Deformability based cell margination--a simple microfluidic design for malaria-infected erythrocyte separation. , 2010, Lab on a chip.