ABSTRACT
Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.
Subject(s)
Neoplasms, Experimental/therapy , Non-alcoholic Fatty Liver Disease/therapy , Nucleic Acids/chemistry , Toll-Like Receptors/agonists , Animals , Antigens/chemistry , Cell Line , Female , Humans , Immunity, Innate , Liver Cirrhosis/pathology , Lymphoma/therapy , Mice , Mice, Inbred C57BL , Nanomedicine/methods , Nanoparticles/chemistry , Nucleic Acid Conformation , Nucleic Acids/therapeutic use , Oligonucleotides/therapeutic useABSTRACT
Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in bloodso-called circulating tumor cells (CTCs)may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.
Subject(s)
Carbocyanines/chemistry , DNA, Antisense/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Neoplastic Cells, Circulating/chemistry , Base Sequence , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Breast Neoplasms/blood , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cadherins/genetics , Cadherins/metabolism , Carbocyanines/metabolism , Cell Line, Tumor , DNA, Antisense/genetics , DNA, Antisense/metabolism , Female , Fibronectins/genetics , Fibronectins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Nanotechnology/methods , Neoplastic Cells, Circulating/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transplantation, Heterologous , Vimentin/genetics , Vimentin/metabolism , Red Fluorescent ProteinABSTRACT
The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.
Subject(s)
Cortactin/chemistry , Proto-Oncogene Proteins c-abl/chemistry , Amino Acid Substitution , Animals , Cells, Cultured , Cortactin/genetics , Cortactin/metabolism , Crystallography, X-Ray , Fibroblasts , Humans , Mice , Mice, Knockout , Mutation, Missense , Protein Binding , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Structure-Activity Relationship , src Homology DomainsABSTRACT
There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are "pro-fluorescent" biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3',6'-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for tetraserine motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range and prefers this sequence to simple monosaccharides by >10,000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal, and TIRF microscopy, but fluoresces brightly within the Ser-Ser-Pro-Gly-Ser-Ser-rich cell interior. These results suggest that current efforts to identify optimal serine-rich sequences for RhoBo will allow it to function effectively as a selective small-molecule label for appropriately tagged proteins either upon or within living cells.
