2 Networking Center on Bioengineering Biomaterials and Nanomedicine CIBERBBN Spain 3 Sant Pau and Josep Carreras Research Institute Hospital de Santa Creu i Sant Pau Barcelona Spain INTRODUCTION ID: 1045538
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1. 1 Nucleic Acids Chemistry Group, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain2 Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain 3 Sant Pau and Josep Carreras Research Institute, Hospital de Santa Creu i Sant Pau, Barcelona, SpainINTRODUCTIONOBJECTIVEMETHODOLOGYFig. 1 Formation of DNA origami:T22GFPH6 complex.a. Conjugation of oligo15T to T22GFPH6 b. Conjugation of T22GFPH6 to DNA origami with oligo15A capture staplesa. b.RESULTSThese results confirm the huge potential of the DNA origami-T22GFPH6 complexes as therapeutic carriers providing nanoscale precision scaffolds, with hundreds of positions ready to be loaded with therapeutic molecules combined with high affinity vectors for cell receptors such as CXCR4 specific, overexpressed in cancer stem cells, opening promising possibilities in cancer treatment. CONCLUSIONFig. 2 Characterization of DNA origami-T22GFPH6 complex.a. On the left SYBRGreen-stained 1% agarose gel image and on the right fluorescence channels of the step-by-step assembly of DNA origami-protein complex. Lane 1: Marker. Lane 2: M13 genome DNA. Lane 3: rectangular DNA origami template with capture strands. Lane 4: DNA origami-T22GFPH6 complexes. Lane 5: T22GFPH6oligo15T. Lane 6: Staples. b. Hydrodynamic sizes of DNA origami template (blue) and DNA origami:T22GFPH6 complex (red). c. AFM images of different T22GFPH6 conformations on the DNA origami (from left to right; unmodified DNA origami, DNA origami:T22GFPH6 monomer, DNA origami:T22GFPH6 pentamer and DNA origami:T22GFPH6 octamer). d. AFM images showing T22GFPH6oligoT15 bound to the DNA origami at different ratios and yield of T22GFPH6-loading structure (%). The efficiency of T22GFPH6 loading was calculated from AFM images by dividing the number of T22GFPH6- binding structures (highlighted by the blue circle) by the total number of origami assemblies counted.Fig. 3 Biological activity of DNA origami-T22GFPH6 complex. It was studied in two different colorectal cancer cells: SW480 (Dukes' type B) and SW620 (Dukes' type C, overexpression of CXCR4 receptor)a. Cellular internalization of the DNA origami:protein complex in two different cell lines. b. Cytoxic activity of DNA Origami:T22GFPH6 complex loaded with two different antitumoral drugs: FdU and doxorubicin.DNA is an attractive molecular building block to construct nanoscale structures for multiple applications. In addition to their unique structure, the conjugation and chemical modification of DNA nanostructures with other molecules opens unlimited possibilities in the production of functional DNA-based architectures. Recent advances in DNA origami have begun to realize this potential however it is still at the earliest stage and a number of hurdles remain. For therapeutic applications, one of these challenges regards to the internalization of DNA scaffolds into cells for an efficient drug delivery. CXCR4 receptor is a cell surface marker overexpressed in several human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Previously, T22GFPH6 protein has been designed to bind to CXCR4 and penetrate colorectal cancer cells efficiently via CXCR4 specific endocytosis.A DNA origami-T22GFPH6 complex was assembled through Watson and Crick complementary base pairing by means of chemical modifications of both. Moreover, the cellular internalization and the cytotoxic activity of the DNA origami-T22GFPH6 complexes loaded with different prodrugs, which are considered successful agents in the treatment of colorectal cancer, were assayed.A DNA origami-protein complex as a therapeutic nanocarrier for intracellular CXCR4+ cell targetingN. Navarro1,2, U. Unzueta2,3, R. Eritja1,2, C. Fàbrega1,2 a. b.c. d.Fig. 2 Characterization of DNA origami-T22GFPH6 complex.a. On the left SYBRGreen-stained 1% agarose gel image and on the right fluorescence channels of the step-by-step assembly of DNA origami-protein complex. Lane 1: Marker. Lane 2: M13 genome DNA. Lane 3: rectangular DNA origami template with capture strands. Lane 4: DNA origami-T22GFPH6 complexes. Lane 5: T22GFPH6oligo15T. Lane 6: Staples. b. Hydrodynamic sizes of DNA origami template (blue) and DNA origami:T22GFPH6 complex (red). c. AFM images of different T22GFPH6 conformations on the DNA origami (from top to bottom; unmodified DNA origami, DNA origami:T22GFPH6 monomer, DNA origami:T22GFPH6 pentamer and DNA origami:T22GFPH6 octamer). d. AFM images showing T22GFPH6oligoT15 bound to the DNA origami at different ratios and yield of T22GFPH6-loading structure (%). The efficiency of T22GFPH6 loading was calculated from AFM images by dividing the number of T22GFPH6- binding structures (highlighted by the blue circle) by the total number of origami assemblies counted.a. b.Unmodified origamiOrigami:T22GFPH6********Internalization (%)SW480SW620SW480SW620Cell viability (%)***Unmodified origamiT22GFPH6Origami FdUOrigami FdU:T22Origami DoxoOrigami Doxo:T22
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