A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants.

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

The Influence of Reaction Conditions on DNA Multimerization During Isothermal Amplification with Bst exo- DNA Polymerase.

Methods for isothermal amplification of nucleic acids are gained more attention in the last two decades. For isothermal amplification, DNA polymerases with strand displacement activity are required, and Bst exo- is one of the most commonly used polymerases. However, Bst exo- is able to cause nonspecific DNA amplification through multimerization, which leads to a set of undesirable by-products. In this study, circumstances that facilitate DNA multimerization by Bst exo- polymerase have been determined. We found that an essential requirement for multimerization is the presence of short (50-60 bp) DNA duplexes formed through primer extension after annealing on the template or in homo- and heterodimers. The highest multimerization efficiency is observed for Bst 2.0 polymerase in buffers with a high salt concentration and/or in the presence of reducing agents (for example, ß-mercaptoethanol). Multimerization occurs mainly at 55-60 °Ð¡, while specific isothermal amplification is more efficient at 60-65 °Ð¡. The SYBR Green I intercalating dye inhibits multimerization with Bst LF and Bst 2.0 polymerases in concentrations above 0.25×, whereas inhibition with Bst 3.0 polymerase occurs only above 1.25×. The obtained results allow to elaborate accurate and reliable methods for isothermal amplification of nucleic acids.

Effect of metal ions on isothermal amplification with Bst exo- DNA polymerase.

Over the last two decades, the isothermal amplification has become actively used for nucleic acids analysis. To perform isothermal techniques, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most widely used. However, Bst exo- is prone to non-specific DNA synthesis (e.g., DNA multimerization) occurring in the absence of the DNA target of interest. Here, we report on the activity of Bst exo- in the presence of Mg2+, Mn2+, Ca2+, Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ in the model molecular systems which included amplification of circular and linear DNA templates; conditions providing effective and highly specific isothermal amplification were determined. It was found that amplification can proceed not only with Mg2+ but with Mn2+, Ca2+, Cd2+ and Cu2+ depending on the type of Bst exo- polymerase and the buffer. Manganese ions turned out to be the most suitable alternative cofactor, which prevents multimerization in some buffers. Molecular docking simulations showed the highest stability for the quaternary 'polymerase-DNA-triphosphate-cations' complexes containing Mg2+ and Mn2+, and the moderate one for complexes with Ca2+, Cd2+ and Cu2+. The frequency of nucleotide misincorporation increased in the following row: Mg2+ ≈ Mn2+ ≤ Cd2+ < Ca2+ â‰ª Cu2+.

Data on molecular docking simulations of quaternary complexes 'Bst exo- polymerase-DNA-dCTP-metal cations'.

This article reports data related to the research article entitled "Effect of metal ions on isothermal amplification with Bst exo- DNA polymerase" (R.R. Garafutdinov, A.R. Gilvanov, O.Y. Kupova, A.R. Sakhabutdinova, 2020) [1]. Here, the results of molecular simulations of the complexes of Bst exo- DNA polymerase with dCTP triphosphate, double-stranded DNA and divalent metal cations are presented. Energetic parameters, number and type of chemical bonds formed by dCTP with the environment are given.