Synthesis and NMR spectral properties of spin-labelled mRNA5′ cap analogue: a new tool for biochemical studies of cap binding proteins

Abstract

All eukaryotic messenger RNAs (mRNAs) and small nuclear RNAs (snRNAs)comprise a unique chemical structure called a ‘cap’, i.e. 7-methylguanosine linked by a5′,5′ triphosphate bridge to the first transcribed nucleoside. Biophysical studies of interactions between theRNA 5′ terminus and proteins that specifically recognize its structure require suitable chemical capanalogues. For the needs of electron spin resonance spectroscopy, a spin-labelled cap analogue,m7GTP-TEMPO, P1-(7-methylguanosine-5′) P3-(2,2,6,6-tetramethyl-1-piperidinyloxy-4) triphosphate, has been synthesized andfully characterized spectroscopically. The structure has been confirmed byone-dimensional (1D) and 2D nuclear magnetic resonance (NMR), electron spinresonance (ESR) and electrospray ionization mass spectrometry (ESI-MS).Despite the presence of a free radical (TEMPO) in the small molecule, complete1H,13C,and 31P NMR spectra have been acquired that allowed us to assign all these resonances, including theradical moiety. These are the first well resolved NMR spectra of the TEMPO-containingparamagnetic species, directly obtained and analysed without conversion to anN-hydroxylamine derivative.