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

Christina M Birch,Han Wei Hou,Jongyoon Han,Jacquin C Niles

doi:10.1038/srep11347

Abstract

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.

Highlights

Ligands displayed on the surface of infected RBC surface is recognized as an important mechanism mediating severe malaria, a detailed characterization has been impeded by limitations in the technologies available for interrogating the infected RBC surface proteome
We have validated a novel and generalizable I-SELEX strategy that is broadly useful for identifying aptamers, and demonstrated its application to model targets displayed on whole cells and live malaria parasite-infected RBCs
We show that this approach facilitates recovery of a diverse set of high affinity and specific aptamers to either a single target artificially displayed on a cell surface or a complex combination of targets natively displayed on whole cells

Summary

Introduction

Ligands displayed on the surface of infected RBC surface is recognized as an important mechanism mediating severe malaria, a detailed characterization has been impeded by limitations in the technologies available for interrogating the infected RBC surface proteome. This approach likely underestimates and in some instances misrepresents the repertoire of parasite proteins present on the infected RBC surface Specific affinity reagents, such as antibodies, can be powerful for determining the cell surface localization and, through pull-down coupled with mass spectrometry, the identity of target proteins. We have sought to address this challenge by establishing a new strategy for increasing access to diverse sets of nucleic acid aptamer affinity reagents capable of recognizing distinct epitopes on the surface of parasite-infected RBCs. We reason that aptamers could be ideal affinity reagents to develop as they can: (1) bind their targets with high affinities and specificities as antibodies; (2) be produced inexpensively in vitro; and (3) be identified through an in vitro selection process (SELEX)[16,17] using live, whole parasite-infected RBCs as targets. I-SELEX should be broadly useful for other SELEX applications using bead-immobilized or live, unmodified whole cell targets

Methods

Results

Conclusion