In Vitro Selection of RNA Aptamers That Bind to Cell Adhesion Receptors of Trypanosoma cruzi and Inhibit Cell Invasion

Abstract

Trypanosoma cruzi causing Chagas' disease needs to invade host cells to complete its life cycle. Macromolecules on host cell surfaces such as laminin, thrombospondin, heparan sulfate, and fibronectin are believed to be important in mediating parasite-host cell adhesions and in the invasion process of the host cell by the parasite. The SELEX technique (systematic evolution of ligands byexponential enrichment) was used to evolve nuclease-resistant RNA ligands (aptamer = to fit) that bind with affinities of 40–400 nm to parasite receptors for the host cell matrix molecules laminin, fibronectin, thrombospondin, and heparan sulfate. After eight consecutive rounds of in vitro selection four classes of RNA aptamers based on structural similarities were isolated and sequenced. All members of each class shared a common sequence motif and competed with the respective host cell matrix molecule that was used for displacement during the selection procedure. RNA pools following seven and eight selection rounds as well as individual aptamers sharing consensus motifs were active in inhibiting invasion of LLC-MK2 monkey kidney cells by T. cruzi in vitro. Trypanosoma cruzi causing Chagas' disease needs to invade host cells to complete its life cycle. Macromolecules on host cell surfaces such as laminin, thrombospondin, heparan sulfate, and fibronectin are believed to be important in mediating parasite-host cell adhesions and in the invasion process of the host cell by the parasite. The SELEX technique (systematic evolution of ligands byexponential enrichment) was used to evolve nuclease-resistant RNA ligands (aptamer = to fit) that bind with affinities of 40–400 nm to parasite receptors for the host cell matrix molecules laminin, fibronectin, thrombospondin, and heparan sulfate. After eight consecutive rounds of in vitro selection four classes of RNA aptamers based on structural similarities were isolated and sequenced. All members of each class shared a common sequence motif and competed with the respective host cell matrix molecule that was used for displacement during the selection procedure. RNA pools following seven and eight selection rounds as well as individual aptamers sharing consensus motifs were active in inhibiting invasion of LLC-MK2 monkey kidney cells by T. cruzi in vitro. Trypanosoma cruzi, the protozoan causing Chagas' disease, must invade host cells to complete its biological cycle. The parasite exists in three developmental forms. Epimastigotes extracellularly multiply in the gut of insect vectors. The mammalian stage of the parasite life cycle includes amastigotes, an intracellular form that replicates, an epimastigote-like form and trypomastigotes, released to the extracellular milieu to invade new cells or biting insects, thus propagating the infection (1Brener Z. Annu. Rev. Microbiol. 1973; 27: 347-381Crossref PubMed Scopus (486) Google Scholar, 2Almeida-de-Faria M. Freymüller E. Colli W. Alves M.J.M. Exp. Parasitol. 1999; 92: 263-274Crossref PubMed Scopus (76) Google Scholar).To contact and invade a mammalian host cell trypomastigotes must cross a network of extracellular matrix proteins on the of cells of the and for in the of cell infection by was by of Trypanosoma and W. and Scholar, Colli W. Alves M.J.M. PubMed Scopus Google cell matrix molecules such as fibronectin PubMed Scopus Google Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google heparan M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google and proteins of the the cell Colli W. Alves M.J.M. PubMed Scopus Google bind to the parasite and host cell the of host a Colli W. Alves M.J.M. 1999; PubMed Scopus Google and to a of that on cell surfaces of the stage and to and on host cells Colli W. Alves M.J.M. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google an sequence with the that to the Annu. Rev. Microbiol. PubMed Scopus Google Scholar, W. PubMed Scopus Google and sequence motifs that to bind to proteins of the cell matrix Colli W. Alves M.J.M. PubMed Scopus Google on that a of host cell and parasite T. cruzi to invade an in vitro selection technique was for the of nuclease-resistant RNA aptamers that with host cell matrix molecules for on T. cruzi cell surfaces Alves M.J.M. Colli W. RNA and aptamers as to cell in Scopus Google SELEX used evolution of ligands byexponential used evolution of ligands byexponential (systematic evolution of ligands byexponential enrichment) PubMed Scopus Google Scholar, PubMed Scopus Google an selection that used to ligands that bind with and affinities to a of proteins and cell and PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, and PubMed Scopus Google as well as to such as cell M. PubMed Scopus Google the PubMed Scopus Google Scholar, and W. 92: PubMed Scopus Google and M. 1999; 27: PubMed Scopus Google The of the RNA in the of aptamers as to in in vitro and in W. Google RNA and aptamers to cell in disease in the The biological of such aptamers in in vitro RNA and PubMed Scopus Google Scholar, T. PubMed Scopus Google PubMed Scopus Google RNA aptamers that bind to and infection in the of the SELEX technique for the evolution of that cells infection by nuclease-resistant RNA and ligands are for in and 1999; PubMed Scopus Google and Google SELEX technique was used to evolve nuclease-resistant RNA aptamers that in vitro T. cruzi trypomastigotes and monkey kidney LLC-MK2 host cells and cell invasion by was and were was and by of and Epimastigotes of T. cruzi T. cruzi were by infection of monkey kidney cells as Colli W. 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RNA and the a of a its of of SELEX RNA or or individual aptamers consensus motifs in of was in the of and of and on a RNA with RNA to a of nm was for with trypomastigotes of or with of in the of and of fibronectin to in of selection The were by The and RNA to surfaces were with of selection and or were with of SELEX or individual structural classes in the of in of selection for was in the of The was by the to the = the of for the RNA of to T. cruzi trypomastigotes were for with the SELEX RNA or or individual based on consensus in LLC-MK2 cells to were in a with trypomastigotes in the or of SELEX RNA pools for The was by the trypomastigotes and with The were by with by with The cells were with with for with and with as Colli W. Alves M.J.M. PubMed Scopus Google All were in and of each were the of and cells in cells The were for by the The of cells and trypomastigotes in the of was by with and the of the host cell matrix molecules such as fibronectin, laminin, thrombospondin, and heparan with respective host cell receptors on of T. a of proteins in the extracellular of fibronectin PubMed Scopus Google Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google and heparan M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google are ligands for T. cruzi in the mammalian the of in T. cruzi invasion to of by a Microbiol. 1999; Google and with Exp. Scopus Google Scholar, PubMed Scopus Google fibronectin, laminin, and heparan as important molecules in host cell invasion by T. cruzi that trypomastigotes molecules to cell W. Alves M.J.M. Colli W. Exp. Parasitol. PubMed Scopus Google and biological in vitro. The of the RNA in to the invasion of the host cell by the parasite that T. cruzi to invade host to the of molecules in the as in host cell invasion by the parasite of Trypanosoma and W. and Scholar, Colli W. Alves M.J.M. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google Scholar, M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google Scholar, Annu. Rev. Microbiol. PubMed Scopus Google Scholar, W. PubMed Scopus Google to the of the parasite with cell matrix parasite E. PubMed Scopus Google Scholar, W. Exp. PubMed Scopus Google and host cell proteins such as W. Exp. PubMed Scopus Google M. PubMed Scopus Google or 1999; PubMed Scopus Google receptors to in host cell of the host cell for T. cruzi cell a of receptors and ligands to its with of molecules on the of the respective cell the SELEX new to cell that the of the by the RNA aptamers are to are for in that parasite-host cell be used to that the of to that are for of the life cycle of such as in vitro of ligands a that and invasion of insect cells by was M. PubMed Scopus Google be for with the as the selection of for a of infection by of that and host cells important of cells invasion by to cells to cells of and the respective the host cell matrix molecules in cell invasion by T. cruzi as in are used as ligands by cells Exp. PubMed Scopus Google the in of aptamers by aptamers in the to by with such as or by T. E. PubMed Scopus Google an to as used in as a for Google aptamers that to be in such as T. 1999; PubMed Scopus Google and PubMed Scopus Google and T. M. 1999; PubMed Scopus Google Scholar, M. PubMed Google be to active aptamers in infection by T. cruzi that new to in the of Chagas' Trypanosoma cruzi, the protozoan causing Chagas' disease, must invade host cells to complete its biological cycle. The parasite exists in three developmental forms. Epimastigotes extracellularly multiply in the gut of insect vectors. The mammalian stage of the parasite life cycle includes amastigotes, an intracellular form that replicates, an epimastigote-like form and trypomastigotes, released to the extracellular milieu to invade new cells or biting insects, thus propagating the infection (1Brener Z. Annu. Rev. Microbiol. 1973; 27: 347-381Crossref PubMed Scopus (486) Google Scholar, 2Almeida-de-Faria M. Freymüller E. Colli W. Alves M.J.M. Exp. Parasitol. 1999; 92: 263-274Crossref PubMed Scopus (76) Google contact and invade a mammalian host cell trypomastigotes must cross a network of extracellular matrix proteins on the of cells of the and for in the of cell infection by was by of Trypanosoma and W. and Scholar, Colli W. Alves M.J.M. PubMed Scopus Google cell matrix molecules such as fibronectin PubMed Scopus Google Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google heparan M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google and proteins of the the cell Colli W. Alves M.J.M. PubMed Scopus Google bind to the parasite and host cell the of host a Colli W. Alves M.J.M. 1999; PubMed Scopus Google and to a of that on cell surfaces of the stage and to and on host cells Colli W. Alves M.J.M. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google an sequence with the that to the Annu. Rev. Microbiol. PubMed Scopus Google Scholar, W. PubMed Scopus Google and sequence motifs that to bind to proteins of the cell matrix Colli W. Alves M.J.M. PubMed Scopus Google on that a of host cell and parasite T. cruzi to invade an in vitro selection technique was for the of nuclease-resistant RNA aptamers that with host cell matrix molecules for on T. cruzi cell surfaces Alves M.J.M. Colli W. RNA and aptamers as to cell in Scopus Google The SELEX used evolution of ligands byexponential used evolution of ligands byexponential (systematic evolution of ligands byexponential enrichment) PubMed Scopus Google Scholar, PubMed Scopus Google an selection that used to ligands that bind with and affinities to a of proteins and cell and PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, and PubMed Scopus Google as well as to such as cell M. PubMed Scopus Google the PubMed Scopus Google Scholar, and W. 92: PubMed Scopus Google and M. 1999; 27: PubMed Scopus Google The of the RNA in the of aptamers as to in in vitro and in W. Google evolution of ligands byexponential evolution of ligands byexponential RNA and aptamers to cell in disease in the The biological of such aptamers in in vitro RNA and PubMed Scopus Google Scholar, T. PubMed Scopus Google PubMed Scopus Google RNA aptamers that bind to and infection in the of the SELEX technique for the evolution of that cells infection by nuclease-resistant RNA and ligands are for in and 1999; PubMed Scopus Google and Google The SELEX technique was used to evolve nuclease-resistant RNA aptamers that in vitro T. cruzi trypomastigotes and monkey kidney LLC-MK2 host cells and cell invasion by was and were was and by of and Epimastigotes of T. cruzi T. cruzi were by infection of monkey kidney cells as Colli W. PubMed Scopus Google LLC-MK2 cells with were in of and The trypomastigotes released the cells are three in by and in a of for of of T. cruzi and fibronectin were and The following of with a on by was a nm by PubMed Scopus Google The of the was on a the for of RNA of were in a of rounds of in the of and The used for were and The was and in vitro of the were of RNA in the of of and and and and the to of the Scopus Google The RNA was to a of in the and and to for T. cruzi trypomastigotes, by were in an of nm for host cell matrix molecules and for with of the RNA T. cruzi trypomastigotes and RNA molecules to surfaces were by for with of and for in of a each and fibronectin and and nm The trypomastigotes were by and the RNA molecules was The was and and in the of of seven of SELEX a was to RNA molecules the SELEX that bind to The RNA that by and SELEX seven was for with in a of The of RNA was and used for selection of cell matrix RNA molecules to trypomastigotes as each of the RNA was and The pools were for the of selection an of of of and of and of RNA the RNA was and and in SELEX cycle the of the pools of aptamers that were laminin, fibronectin, heparan sulfate, or as a displacement were with and the and PubMed Scopus Google The were or and on were and were by the The of the were and for sequence RNA were by the M. RNA and the a of a its of of SELEX RNA or or individual aptamers consensus motifs in of was in the of and of and on a RNA with RNA to a of nm was for with trypomastigotes of or with of in the of and of fibronectin to in of selection The were by The and RNA to surfaces were with of selection and or were with of SELEX or individual structural classes in the of in of selection for was in the of The was by the to the = the of for the RNA of to T. cruzi trypomastigotes were for with the SELEX RNA or or individual based on consensus in LLC-MK2 cells to were in a with trypomastigotes in the or of SELEX RNA pools for The was by the trypomastigotes and with The were by with by with The cells were with with for with and with as Colli W. Alves M.J.M. PubMed Scopus Google All were in and of each were the of and cells in cells The were for by the The of cells and trypomastigotes in the of was by with and was and were was and by was and were was and by of and Epimastigotes of T. cruzi T. cruzi were by infection of monkey kidney cells as Colli W. PubMed Scopus Google LLC-MK2 cells with were in of and The trypomastigotes released the cells are three in by and in a of T. cruzi were by infection of monkey kidney cells as Colli W. 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Alves M.J.M. PubMed Scopus Google All were in and of each were the of and cells in cells The were for by the The of cells and trypomastigotes in the of was by with and to T. cruzi trypomastigotes were for with the SELEX RNA or or individual based on consensus in LLC-MK2 cells to were in a with trypomastigotes in the or of SELEX RNA pools for The was by the trypomastigotes and with The were by with by with The cells were with with for with and with as Colli W. Alves M.J.M. PubMed Scopus Google All were in and of each were the of and cells in cells The were for by the The of cells and trypomastigotes in the of was by with and the of the host cell matrix molecules such as fibronectin, laminin, thrombospondin, and heparan with respective host cell receptors on of T. a of proteins in the extracellular of fibronectin PubMed Scopus Google Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google and heparan M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google are ligands for T. cruzi in the mammalian the of in T. cruzi invasion to of by a Microbiol. 1999; Google and with Exp. Scopus Google Scholar, PubMed Scopus Google fibronectin, laminin, and heparan as important molecules in host cell invasion by T. cruzi that trypomastigotes molecules to cell W. Alves M.J.M. Colli W. Exp. Parasitol. PubMed Scopus Google and biological in vitro. The of the RNA in to the invasion of the host cell by the parasite that T. cruzi to invade host to the of molecules in the as in host cell invasion by the parasite of Trypanosoma and W. and Scholar, Colli W. Alves M.J.M. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google Scholar, M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google Scholar, Annu. Rev. Microbiol. PubMed Scopus Google Scholar, W. PubMed Scopus Google to the of the parasite with cell matrix parasite E. PubMed Scopus Google Scholar, W. Exp. PubMed Scopus Google and host cell proteins such as W. Exp. PubMed Scopus Google M. PubMed Scopus Google or 1999; PubMed Scopus Google receptors to in host cell of the host cell for T. cruzi cell a of receptors and ligands to its with of molecules on the of the respective cell the SELEX new to cell that the of the by the RNA aptamers are to are for in that parasite-host cell be used to that the of to that are for of the life cycle of such as in vitro of ligands a that and invasion of insect cells by was M. PubMed Scopus Google be for with the as the selection of for a of infection by of that and host cells important of cells invasion by to cells to cells of and the respective the host cell matrix molecules in cell invasion by T. cruzi as in are used as ligands by cells Exp. PubMed Scopus Google the in of aptamers by aptamers in the to by with such as or by T. E. PubMed Scopus Google an to as used in as a for Google aptamers that to be in such as T. 1999; PubMed Scopus Google and PubMed Scopus Google and T. M. 1999; PubMed Scopus Google Scholar, M. PubMed Google be to active aptamers in infection by T. cruzi that new to in the of Chagas' The the of the host cell matrix molecules such as fibronectin, laminin, thrombospondin, and heparan with respective host cell receptors on of T. a of proteins in the extracellular of fibronectin PubMed Scopus Google Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google and heparan M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google are ligands for T. cruzi in the mammalian the of in T. cruzi invasion to of by a Microbiol. 1999; Google and with Exp. Scopus Google Scholar, PubMed Scopus Google fibronectin, laminin, and heparan as important molecules in host cell invasion by T. cruzi that trypomastigotes molecules to cell W. Alves M.J.M. Colli W. Exp. Parasitol. PubMed Scopus Google and biological in vitro. The of the RNA in to the invasion of the host cell by the parasite that T. cruzi to invade host to the of molecules in the as in host cell invasion by the parasite of Trypanosoma and W. and Scholar, Colli W. Alves M.J.M. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Colli W. Alves M.J.M. Parasitol. PubMed Scopus Google Scholar, Colli W. Alves M.J.M. 1999; PubMed Scopus Google Scholar, M. E. Parasitol. Scopus Google Scholar, W. PubMed Scopus Google Scholar, Annu. Rev. Microbiol. PubMed Scopus Google Scholar, W. PubMed Scopus Google to the of the parasite with cell matrix parasite E. PubMed Scopus Google Scholar, W. Exp. PubMed Scopus Google and host cell proteins such as W. Exp. PubMed Scopus Google M. PubMed Scopus Google or 1999; PubMed Scopus Google receptors to in host cell of the host cell for T. cruzi cell a of receptors and ligands to its with of molecules on the of the respective cell the SELEX new to cell that the of the by the RNA aptamers are to are for in that parasite-host cell be used to that the of to that are for of the life cycle of such as in vitro of ligands a that and invasion of insect cells by was M. PubMed Scopus Google be for with the as the selection of for a of infection by The of that and host cells important of cells invasion by to cells to cells of and the respective the host cell matrix molecules in cell invasion by T. cruzi as in are used as ligands by cells Exp. PubMed Scopus Google the in of aptamers by aptamers in the to by with such as or by T. E. PubMed Scopus Google an to as used in as a for Google aptamers that to be in such as T. 1999; PubMed Scopus Google and PubMed Scopus Google and T. M. 1999; PubMed Scopus Google Scholar, M. 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