ABSTRACT
Drug discovery is challenged by ineffectiveness of drugs against variable and evolving diseases, and adverse effects due to poor selectivity. We describe a robust platform which potentially addresses these limitations. The platform enables rapid discovery of DNA oligonucleotides evolved in vitro for exerting specific and selective biological responses in target cells. The process operates without a priori target knowledge (mutations, biomarkers, etc). We report the discovery of oligonucleotides with direct, selective cytotoxicity towards cell lines, as well as patient-derived solid and hematological tumors. A specific oligonucleotide termed E8, induced selective apoptosis in triple-negative breast cancer (TNBC) cells. Polyethylene glycol-modified E8 exhibited favorable biodistribution in animals, persisting in tumors up to 48-hours after injection. E8 inhibited tumors by 50% within 10 days of treatment in patient-derived xenograft mice, and was effective in ex vivo organ cultures from chemotherapy-resistant TNBC patients. These findings highlight a drug discovery model which is target-tailored and on-demand.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Oligodeoxyribonucleotides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Base Sequence , Cell Line, Tumor , Cells, Cultured , Disease Models, Animal , Drug Discovery/methods , Drug Screening Assays, Antitumor , High-Throughput Nucleotide Sequencing , Humans , Mice , Models, Molecular , Molecular Conformation , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/therapeutic use , Structure-Activity Relationship , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
Discriminating between the mRNA and protein outputs of each of the alleles of an endogenous gene in intact cells, is a difficult task. To examine endogenous transcripts originating from a specific allele, we applied Central Dogma tagging (CD-tagging), which is based on a tag insertion into an endogenous gene by creation of a new exon. Previously, CD-tagging was used to tag endogenous proteins. Here we developed a CD-tagging-MS2 approach in which two tags were inserted in tandem; a fluorescent protein tag in conjunction with the mRNA MS2 tag used for tagging mRNAs in cells. A cell clone library of CD-tagged-MS2 genes was generated, and protein and mRNA distributions were examined and characterized in single cells. Taking advantage of having one allele tagged, we demonstrate how the transcriptional activity of all alleles, tagged and untagged, can be identified using single molecule RNA fluorescence in situ hybridization (smFISH). Allele-specific mRNA expression and localization were quantified under normal and stress conditions. The latter generate cytoplasmic stress granules (SGs) that can store mRNAs, and the distribution of the mRNAs within and outside of the SGs was measured. Altogether, CD-tagging-MS2 is a robust and inexpensive approach for direct simultaneous detection of an endogenous mRNA and its translated protein product in the same cell.
ABSTRACT
The kinetic aspects of RNA polymerase II as it transcribes mRNA have been revealed over the past decade by use of live-cell imaging and kinetic analyses. It is now possible to visualize polymerase molecules in action, and most importantly to detect and follow the mRNA product as it is generated in real time on active genes. Questions such as the speed at which mRNAs are transcribed or the number of polymerases running along a particular gene can be addressed at high temporal resolution. These kinetic studies highlight the tight regulation that genes encounter when moving between active and inactive states, and ultimately will shed light on the kinetic aspects of transcription of genes under perturbed states. The scientific pathway along which these findings were unearthed begins with the imaging of the action of hundreds of genes working in concert in fixed cells. The state of the art has reached the capability of analyzing the transcription of single alleles in living mammalian cells.
