Provided herein is a bacterial delivery platform that harnesses mechanisms unique to Salmonella to intracellularly deliver protein-based drugs.

A host cell for protein expression having a lower expression level of a gene, as compared to a wild-type cell, wherein the gene is selected from HDAC8, Dab2, Caspase3, Sys1, Ergic3, Grasp, Trim 23, or a combination thereof. The host cells are CHO cells. The lower expression level of the gene results from RNA interference, which may be achieved by transfecting a vector that contains an shRNA targeting the gene.

Methods, systems, and devices are disclosed for intracellular payload delivery by nanomotor structures. In some aspects, a nanomotor for intracellular payload delivery includes an asymmetric body having a concave cavity at one end of the nanowire body; a functionalization layer on an outer surface of the nanowire body; and a payload substance coupled to the nanomotor by the functionalization layer in a biologically active conformation, wherein the payload substance is attached to a portion of the functionalization layer or at least partially encapsulated within the functionalization layer, in which the nanomotor is operable to propel in a biological medium and into an intracellular region of a living cell to initiate an interaction of the biologically active payload substance with an intracellular constituent of the living cell.

The invention described herein features infrared fluorescent protease reporters (iProteases) and methods of use thereof. The iProteases can be used in in vivo and in vitro assays to detect protease activity and disease states associated with protease activity. In a still further embodiment, the present invention provides a kit comprising any of the above described polynucleotides. In a further aspect, the present invention provides a method of in vivo optical imaging. In a still further embodiment, the in vivo imaging is performed in a living animal. In a further aspect, the present invention provides a method of detecting protease activity, the method comprising expressing a polypeptide according to any of those described above in a cell.

Provided is a system for drug delivery comprising a modified poly (amidoamine) (PAMAM) comprising a photo-cleavable compound bound to one or more active agents to form a nanocomplex. Also provided is a method of treating a subject comprising administering the PAMAM nanocomplex and irradiating the nanocomplex at the target site with a light source. In particular, the modified PAMAM is a DEACM-modified PAMAM or a BODIPY-modified PAMAM. Also provided is a method for screening for PAMAM-coumarin-active agent nanocomplexes.

Methods and constructs are provided for controlling processes in live animals, plants or microbes via genetically engineered near-infrared light-activated or light-inactivated proteins including chimeras including the photosensory modules of bacteriohytochromes and output modules that possess enzymatic activity and/or ability to bind to DNA, RNA, protein, or small molecules. DNA encoding these proteins are introduced as genes into live animals, plants or microbes, where their activities can be turned on by near-infrared light, controlled by the intensity of light, and turned off by near-infrared light of a different wavelength than the activating light. These proteins can regulate diverse cellular processes with high spatial and temporal precision, in a nontoxic manner, often using external light sources. For example, near-infrared light-activated proteins possessing nucleotidyl cyclase, protein kinase, protease, DNA-binding and RNA-binding activities are useful to control signal transduction, cell apoptosis, proliferation, adhesion, differentiation and other cell processes.

The invention described herein features infrared fluorescent protease reporters (iProteases) and methods of use thereof. The iProteases can be used in in vivo and in vitro assays to detect protease activity and disease states associated with protease activity. In a still further embodiment, the present invention provides a kit comprising any of the above described polynucleotides. In a further aspect, the present invention provides a method of in vivo optical imaging. In a still further embodiment, the in vivo imaging is performed in a living animal. In a further aspect, the present invention provides a method of detecting protease activity, the method comprising expressing a polypeptide according to any of those described above in a cell.

Methods and constructs are provided for controlling processes in live animals, plants or microbes via genetically engineered near-infrared light-activated or light-inactivated proteins including chimeras including the photosensory modules of bacteriohytochromes and output modules that possess enzymatic activity and/or ability to bind to DNA, RNA, protein, or small molecules. DNA encoding these proteins are introduced as genes into live animals, plants or microbes, where their activities can be turned on by near-infrared light, controlled by the intensity of light, and turned off by near-infrared light of a different wavelength than the activating light. These proteins can regulate diverse cellular processes with high spatial and temporal precision, in a nontoxic manner, often using external light sources. For example, near-infrared light-activated proteins possessing nucleotidyl cyclase, protein kinase, protease, DNA-binding and RNA-binding activities are useful to control signal transduction, cell apoptosis, proliferation, adhesion, differentiation and other cell processes.

A method for in vitro transcription of a DNA template into RNA includes providing a mixture containing a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, and a recombinant RNA polymerase, and incubating the reaction mixture at from about 25 C. to about 40 C. for from about 1 hour to about 12 hours thereby producing the RNA. A method for in vitro transcription includes providing a DNA template and a cap analogue that binds to 1 and/or +1 nucleotides of promoter for in vitro transcription, thus producing more full length mRNAs, allowing for more flexibility on the choice of first mRNA base, and providing+2 position open for custom sequence.

Disclosed herein include methods, compositions, and kits suitable for use in programmable cell death control. Compositions (e.g., nucleic acid compositions, synthetic protein circuits) provided herein can comprise one or more apoptosis polypeptides; and/or one or more pyroptosis polypeptides; and/or one or more input polypeptides. In some embodiments, expression of a synthetic protein circuit of the disclosure can induce, e.g., apoptosis and/or pyroptosis in a cell.