Fluorescence-Based Methods for the Study of Biomolecular Interactions

Biomolecular interactions are key players in biological processes and thus a fundamentally important aspect of life sciences. Fluorescence techniques have become some of the most powerful and widely applied methods to investigate and understand these processes. In vitro fluorescence approaches offer the potential for real-time detection of extra- and intracellular molecular interactions, which constitutes a major advantage over other approaches. This has been used for a variety of protein-protein, protein-nucleic acid, and protein-membrane interactions, which play crucial roles in the regulation of cellular processes.

This collection gives an overview of various fluorescence-based techniques and applications for the study of biomolecular interactions and broadens our understanding of cellular processes.

Reiser et al.¹ describe a high-throughput method for analyzing cellular kinetics from individual cells via automated image acquisition and processing (Live Cell Imaging on Single Cell Arrays [LISCA]). Using this technique, fluorescent reporters provide a time-resolved readout of various independent biological processes such as single cell gene expression after transfection, cellular response in signaling cascades, apoptotic events, or stem cell differentiation. The authors perform imaging in isolated cell arrays using scanning time-lapse microscopy, leading to hundreds of single cell trajectories and enough data for statistical analysis. Single cell fluorescence time traces will help researchers better understand gene delivery mechanisms and resolve cellular heterogeneity—a hallmark of complex biological systems normally dictated by intrinsic fluctuations.

Konate et al.² report a biophysical method to characterize the interaction, perturbation, and/or permeabilization of membrane interacting peptides. In their protocol, they precisely describe the different steps to formulate and purify liposomes and to characterize fluorescence leakage. The method is applied with a cell-penetrating peptide (CPP) called WRAP in association with a small interfering RNA (siRNA) or combined with a potent therapeutic peptide (iCAL36) and is compared to the well-known CPP, penetratin. This technique presents multiple advantages. It is fast and simple to implement. It allows tight control of the lipid membrane composition to mimic different types of cell membranes. The fluorophores can be adapted depending on the peptide of interest and its membrane leakage properties. Finally, the sample is extremely stable after preparation. The versatility of the technique makes it a useful approach to probe various biological questions regarding cell damage or drug delivery.

The presence of phosphatidylserine (PS) in mammalian cell membranes is important in different signaling processes such as cell apoptosis or in the recruitment of proteins for membrane repair. The transfer and changes in PS distribution at the surface of plasma membranes are a key question that Ikhlef et al.³ tackle with their article. They describe a protocol to measure in real time PS and PI-4-P lipid transport and exchange by the recombinant yeast protein Osh6p. The method utilizes fluorescent sensors to detect whether a lipid transfer protein (LTP) extracts or transfers lipids in artificial membranes along a gradient between lipid vesicles. This protocol enables work with unlabeled lipids to allow the correct binding of lipids to the LTP. In addition, a better time resolution is achieved in comparison to conventional methods, such as radioactivity-based assays or mass spectrometry. This method will help researchers better understand the extraction and exchange of lipids between different organelles and membranes.

Förster resonance energy transfer (FRET) is a powerful method to study biomolecular systems. This technique can be applied to single molecules (e.g., smFRET) and allows the measure of absolute distances within biomolecules. Abdelhamid et al.⁴ present a step-by-step protocol on how to perform smfBox, a recently developed confocal setup capable of measuring the FRET efficiency between two dyes on freely diffusing single molecules. They show how to implement and use the smFRET technique from sample preparation to the instrument setup, alignment, data acquisition, and analysis. Moreover, they describe how to determine the correction factors required for accurate FRET-derived distance measurements. Here, the method is applied to freely diffusing DNA duplexes and can be extended to a broad range of applications such as the study of protein conformational changes or protein-DNA interactions.

Biological processes involve very diverse and numerous biomolecular interactions that leave ample space for the development of new fluorescence-based assays or sensors. This collection offers researchers an overview of the innovations and developments in fluorescence-based methods. Each article sheds light on a different fluorescence-based method, highlighting how important and versatile fluorescent assays have become. The collection describes these methods and thus encourages the wide-spread usage of them among different groups and research areas.