Abstract: One or more new variant(s) of luciferases and fungal luciferin biosynthesis enzymes, nucleic acids able to encode these enzymes, and proteins able to catalyze certain stages of the fungal luciferin biosynthesis are provided herein. More specifically one or more bioluminescent enzymes, and in particular one or more hispidin hydroxylases and luciferases, and functional fragments thereof and nucleic acids encoding them are disclosed.

Abstract: Provided herein are bioluminescent polypeptides and compositions and methods for the assembly of a tripartite or multipartite bioluminescent complex. In particular embodiments, a bioluminescent complex is formed upon the interaction of three or more peptide and/or polypeptide components.

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

Abstract: Provided herein are compositions and systems comprising complementation-based tags and reporters for labeling and detection of targets by luminescence and a second modality (e.g., fluorescence), and methods of use thereof. In particular, tags are provided comprising the fusion of a first component of bioluminescent complex and a first component of modified dehalogenase complex, reporters are provided comprising the second component of bioluminescent complex and the second component of modified dehalogenase complex, and systems and methods are provided comprising the tags and reporters herein for dual-modality labeling and detection of targets.

Abstract: Provided herein are compositions and systems comprising a DNA polymerase domain, a thioredoxin binding domain (TBD), and thioredoxin (TRX), wherein one or both of the TRX and TBD are fused or otherwise conjugated to the DNA polymerase domain. A TRX or TBD may also be provided in a system herein as a separate entity (e.g., a binary system). The DNA polymerase/TBD/TRX compositions and systems herein are engineered to reduce stutter and/or to produce fewer stutter artifacts. Kits comprising the DNA polymerase/TBD/TRX compositions and systems herein and methods of use thereof are also within the scope herein.

Abstract: Provided herein are peptide and polypeptide sequences that structurally assemble to form active, modified dehalogenase structures capable of binding to a haloalkyl ligand. In particular, provided herein are split dehalogenase variants that assemble through structural complementation into active dehalogenase complexes, and systems and methods of use thereof.

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

Abstract: Provided herein modified dehalogenases have extended surface loop regions that provide a location for internal fusion insertions and modulate binding interaction and activation of environmentally-sensitive chemistries.

Abstract: Provided herein are circularly-permuted (cp) dehalogenase variants that are capable of covalently binding to a haloalkyl ligand. In particular cp dehalogenase variants and peptides and polypeptides comprising split versions thereof that structurally assemble to form an active dehalogenase complexes are provided.

Abstract: Provided herein are systems and methods for characterizing target/ligand engagement. In particular, luciferase-labeled polypeptide targets are used to detect or quantify target/ligand engagement (e.g., within a cell or cell lysate).

Abstract: Provided herein are enhanced luciferase enzymes for use with thermostable luciferin analogs for bioluminescent assays. In particular, the present disclosure provides compositions, assays, and methods for performing a bioluminescent assay using enhanced, high-activity luciferase enzymes compatible with thermostable luciferins, such as 5,5-disubstituted luciferin analogs.

Abstract: Provided herein are systems and methods for characterizing target/ligand engagement. In particular, luciferase-labeled polypeptide targets are used to detect or quantify target/ligand engagement (e.g., within a cell or cell lysate).

Abstract: Provided herein are enhanced luciferase enzymes for use with thermostable luciferin analogs for bioluminescent assays. In particular, the present disclosure provides compositions, assays, and methods for performing a bioluminescent assay using enhanced, high-activity luciferase enzymes compatible with thermostable luciferins, such as 5,5-disubstituted luciferin analogs.

Abstract: 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.

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

Abstract: Provided herein are systems and methods for characterizing target/ligand engagement. In particular, luciferase-labeled polypeptide targets are used to detect or quantify target/ligand engagement (e.g., within a cell or cell lysate).

Abstract: 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.

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

Abstract: Provided herein are substantially non-luminescent peptide/polypeptide tags that are inserted internally within a protein of interest or between N-terminal and C-terminal peptides/polypeptides. Interaction of the internally-inserted tag with a complement polypeptide/peptide that is also substantially non-luminescent results in the formation a bioluminescent reporter complex.

Abstract: 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.