A NanoLuc-Based Transactivation Assay in Plants.

Protein-DNA interactions are determinant of the regulation of gene expression in living organisms. Luminescence studies have been used in a wide range of techniques to identify how gene transcription can be regulated by proteins such as transcription factors (TFs). Despite the great advances in the use of luciferases as reporters in the performance of this mechanism, some of them still have disadvantages that have been tried to be solved by the generation of new luciferases that induce a more stable and perfectly visualizable reaction. NanoLuc is a recently described luciferase that has been characterized by its efficient, stable, and powerful luminescence. These qualities have been considered to create a new and efficient reporter system to detect protein-DNA interactions. In this chapter, we take advantage of NanoLuc and describe its use in a reliable procedure to detect protein-DNA interactions in Nicotiana benthamiana extracts and entire leaves.

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity.

Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model.

New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol.