N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

Abstract

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap−protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap−protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Keywords

• mRNA cap
• 7-methylguanosine
• DcpS
• eIF4E
• fluorescent probes
• MST