The present disclosure relates, in general, to a fusion protein construct comprising RNase L and a split reporter system, and methods of using the reporter for detecting 2′-5′ linked oligoadenylates (2-5A) and double stranded RNA in vivo.

The present disclosure relates, in general, to a fusion protein construct comprising RNase L and a split reporter system, and methods of using the reporter for detecting 2′-5′ linked oligoadenylates (2-5A) and double stranded RNA in vivo.

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

A polynucleotide encoding a modified luciferase polypeptide. The modified luciferase polypeptide has at least 60% amino acid sequence identity to a wild-type Oplophorus luciferase and includes at least one amino acid substitution at a position corresponding to an amino acid in a wild-type Oplophorus luciferase of SEQ ID NO:1. The modified luciferase polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the wild-type Oplophorus luciferase.

A polynucleotide encoding a modified luciferase polypeptide. The modified luciferase polypeptide has at least 60% amino acid sequence identity to a wild-type Oplophorus luciferase and includes at least one amino acid substitution at a position corresponding to an amino acid in a wild-type Oplophorus luciferase of SEQ ID NO:1. The modified luciferase polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the wild-type Oplophorus luciferase.

A method of conducting an enzymatic reaction assay involving adenosine 5′-monophosphate (AMP) comprising the steps of reacting one or more compounds and producing AMP, deaminating the AMP to produce IMP, and oxidating the IMP by NAD+ to produce XMP and NADH.

In some aspects, the invention provides methods of identifying, detecting, and/or measuring protein-protein interactions. In some aspects, the invention provides methods of identifying and/or characterizing modulators of protein-protein interactions. In some aspects, the invention provides methods of identifying and/or characterizing modulators of protein activity, wherein the methods are based at least in part on measuring interaction between a chaperone and client protein. In some aspects, the invention provides methods for identifying and/or characterizing compounds and/or for assessing compound specificity, wherein the methods are based at least in part on measuring interaction between a chaperone and client protein. In some embodiments, a client protein is a kinase. In some embodiments, a compound is a kinase inhibitor. In some aspects, the invention provides methods of profiling kinase inhibitor specificity. In some aspects, the invention provides assay systems and/or reagents useful for performing one or more of the inventive methods. In some aspects, the invention provides newly identified targets of a variety of kinase inhibitors. In some aspect, the invention provides methods of inhibiting kinases identified herein as targets of certain kinase inhibitors. In some aspects, the invention provides methods of treating a disease, e.g., cancer, by inhibiting one or more kinase(s) newly identified as targets of certain kinase inhibitors.

In some aspects, the invention provides methods of identifying, detecting, and/or measuring protein-protein interactions. In some aspects, the invention provides methods of identifying and/or characterizing modulators of protein-protein interactions. In some aspects, the invention provides methods of identifying and/or characterizing modulators of protein activity, wherein the methods are based at least in part on measuring interaction between a chaperone and client protein. In some aspects, the invention provides methods for identifying and/or characterizing compounds and/or for assessing compound specificity, wherein the methods are based at least in part on measuring interaction between a chaperone and client protein. In some embodiments, a client protein is a kinase. In some embodiments, a compound is a kinase inhibitor. In some aspects, the invention provides methods of profiling kinase inhibitor specificity. In some aspects, the invention provides assay systems and/or reagents useful for performing one or more of the inventive methods. In some aspects, the invention provides newly identified targets of a variety of kinase inhibitors. In some aspect, the invention provides methods of inhibiting kinases identified herein as targets of certain kinase inhibitors. In some aspects, the invention provides methods of treating a disease, e.g., cancer, by inhibiting one or more kinase(s) newly identified as targets of certain kinase inhibitors.

Provided herein are compositions and methods for monitoring the movement of analytes and/or cellular components across biological membranes (e.g., cell surface internalization). In particular, reporter constructs are provided, the transmembrane movement of which (e.g., by endocytosis) is monitored by methods described herein.