Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies

Abstract

RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (∼25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific 13C/15N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling and selective biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10 %) that drop precipitously for oligonucleotides larger than 50 nt; de novo pyrimidine biosynthesis of NTPs, also a robust technique with modest yields of up to 45%, comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1′ and C5′ and the pyrimidine nucleobase C2, C4, C5 or C6; the method of biomass-produced uniformly- or selectively-labeled NTPs suffers from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of 13C-13C couplings. In contrast, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places 13C/15N labels in the pyrimidine nucleobase (either 15N1, 15N3, 13C2, 13C4, 13C5, or 13C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt, and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full l...

Characterizing RNA Excited States Using NMR Relaxation Dispersion

“…It is important to ensure that all running buffers, reagents, glassware, and equipment used are RNAse-free. Protocols for preparation of uniformly 13 C/ 15 N labeled RNA samples are described elsewhere (Easton et al, 2010; Petrov et al, 2013; Alvarado et al, 2014). …”

Chemo-enzymatic labeling for rapid assignment of RNA molecules

“…Recently we have published methods for the chemo-enzymatic synthesis of pyrimidine and purine nucleotides [3,4,9]. Compared to previously published techniques, our method offers a substantial improvement [72][73][78][79][80][81][82][83].…”

“…A detailed protocol for the synthesis of pyrimidine nucleotides has been previously published [4]. In an attempt to make the process as streamlined as possible, we developed His-tagged versions of proteins that are prohibitively expensive or commercially unavailable [95].…”

“…Addressing this discrepancy is therefore important to further our understanding of the variety of roles RNA molecules play. Nuclear magnetic resonance (NMR) spectroscopy has made fundamental contributions to this end [3][4][5][6][7][8]. In particular, recent developments in the synthesis of site-specifically labeled ribonucleotides [3,4,[8][9][10], when paired with recent advancements in various NMR spectroscopic techniques [11][12][13][14][15][16][17], promise to position NMR as an even more effective tool for determining high-resolution structures of RNAs.…”

“…Nuclear magnetic resonance (NMR) spectroscopy has made fundamental contributions to this end [3][4][5][6][7][8]. In particular, recent developments in the synthesis of site-specifically labeled ribonucleotides [3,4,[8][9][10], when paired with recent advancements in various NMR spectroscopic techniques [11][12][13][14][15][16][17], promise to position NMR as an even more effective tool for determining high-resolution structures of RNAs. These newer methodologies will complement earlier NMR developments [18][19][20][21][22][23][24] as well as various site-specific selective and segmental labeling for RNAs [25][26][27][28][29][30] and DNAs [31][32][33][34][35][36][37][38][39][40][41][42][43].…”