Synthesis, base pairing and structure studies of geranylated RNA

Abstract

Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium. The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNAGluUUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon–anticodon interaction during ribosome binding.

Synthesis of Geranyl‐2‐Thiouridine‐Modified RNA

“…This hydrophobic geranyl group has been known to affect codon recognition and frameshifting during translation (Dumelin et al, 2012). In previous studies, both the 2 -deoxy-and ribo-versions of S-geranyluridine have been synthesized and the geranylated 2-thioT-G or 2-thioU-G pairs showed much stronger thermal stability than the corresponding T-A and other mismatched pairs in the DNA and RNA duplex contexts, which is consistent with the observation that the geranylated tRNA Glu UUC recognizes GAG more efficiently than GAA (Wang et al, 2015;Wang et al, 2016). This unit contributes the synthesis of geranylated 2-thiouridine Trimethylsilyl chloride (purified by redistillation, ࣙ99%, Sigma-Aldrich) 1-O-Acetyl-2,3,5-tri-O-benzoyl β-D-ribofuranose (99.5% pure, ChemGenes) 1,2-Dichloroethane (anhydrous, 99.8%, Sigma-Aldrich) NaCl (aqueous, saturated) Tin(IV) chloride (99.995% trace metals basis, Sigma-Aldrich) Saturated aqueous sodium bicarbonate (NaHCO 3 ) Methylene chloride (dichloromethane, CH 2 Cl 2 ; purity >99.5%, Fluka) Anhydrous magnesium sulfate (MgSO 4 ) Methanol (anhydrous MeOH, Sigma-Aldrich) Sodium methoxide (99.995% trace metals basis, Sigma-Aldrich) DOWEX 50WX8-400 ion-exchange resin (H + form) (Sigma-Aldrich) Ethyl acetate (EtOAc) Ethanol (absolute) N,N-Dimethylformamide (DMF; anhydrous, 99% pure, Sigma-Aldrich) Di-tert-butylsilyl bis(trifluoromethanesulfonate) (97%, Sigma-Aldrich) Imidazole (ACS reagent, ࣙ99%, Sigma-Aldrich) tert-Butyldimethylsilyl chloride (>97%, Sigma-Aldrich) HCl (aqueous, 1.0 M) Anhydrous sodium sulfate (Na 2 SO 4 ) Silica gel (60-Å porosity; 40-to 63-μm particle size; 400 mesh) HF-pyridine (ß30% pyridine/ß70% hydrogen fluoride, Sigma-Aldrich) Pyridine (anhydrous, purity >99%, Sigma-Aldrich) 4,4 -Dimethoxytrityl chloride (95%, Sigma-Aldrich) Triethylamine (>99%, Sigma-Aldrich) N,N-Diisopropylethylamine (DIPEA; 99% pure, Sigma-Aldrich) Geranyl bromide (95%, Sigma-Aldrich) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (ChemGenes Corporation) 250-mL three-neck, round-bottom flasks, oven-dried Magnetic stir bars Reflux apparatus 250-mL one-neck, round-bottom flasks, oven-dried Rotary evaporator Vacuum oil pump 1-, 5-, and 10-mL syringes 1000-mL beakers Filter paper pH paper Lyophilizer 10-, 25-, 50-, and 100-mL round-bottom flasks Thermometer Hot plate Separatory funnels Rubber septum 22 × 457-mm silica gel chromatography columns Additional reagents and equipment for performing thin-layer chromatography (TLC) and column chromatography 3.…”

Escherichia coli tRNA 2‐selenouridine synthase (SelU) converts S2U‐RNA to Se2U‐RNA via S‐geranylated‐intermediate

“…Three model 17‐nucleotide RNA oligomers of the sequence 5′‐GUUGACU X UUAAUCAAC‐3′, where X = S2U ( 1c ), Se2U ( 2c ), or geS2U ( 3c ) (Table ), encoded S2U‐RNA, geS2U‐RNA, and Se2U‐RNA, respectively, were prepared (as described earlier ) to help verify the hypothesis on the SelU‐promoted sequential transformation S2U‐RNA→geS2U‐RNA→Se2U‐RNA. The sequence was selected intentionally as the S2U‐RNA oligomer is iso ‐sequential with the anticodon‐stem‐loop fragment (ASL) of the bacterial tRNA specific for lysine (tRNA Lys ), and the modified units (X) occupy the site corresponding to the wobble position in transfer RNA.…”

“…Three model 17-nucleotide RNA oligomers of the sequence 5 0 -GUUGACU X UUAAUCAAC-3 0 , where X = S2U (1c), Se2U (2c), or geS2U (3c) ( Table 1), encoded S2U-RNA, geS2U-RNA, and Se2U-RNA, respectively, were prepared (as described earlier [10,21,22]) to help verify the hypothesis on the SelUpromoted sequential transformation S2U-RNA? geS2U-RNA?Se2U-RNA.…”

“…There are evidences that the presence of geranyl group at S2U 34 affects codon bias and frameshift during translation [4]. The superior recognition of the 3 0 -G-ending codons over the 3 0 -A-ending codons by S-geranylated tRNA Lys in comparison to S2U-RNAs has been observed in vivo [4], and confirmed by in vitro thermodynamic studies with the use of geS2U-RNA models demonstrating preferable binding of geS2U with G (and not with A) in the complementary RNA duplexes [21,22]. Recent experiments performed in three different media and at three different temperatures with genetically engineered bacteria (with different levels of geranylated tRNA) showed that there is no difference in bacteria growth [6].…”

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function

“…Both crystallographic and computational studies of the τ 5 mU 34 -modified ASL of mitochondrial tRNA Leu UAA have revealed stabilized hydrogen bonding interactions between the sulfonic acid and secondary amine moieties of the modification and nucleosides U 33 , A 35 and A 36 , as well as adoption of a non-Watson-Crick U 34 •G 3 geometry thought to enhance stacking [120,153]. Likewise, 2-thiouridine geranylation (Figure 2b) in bacteria facilitates tRNA Glu UUC and tRNA Lys UUU formation of stable Watson-Crick U 34 •G 3 wobble base pairs at the anticodon-codon interface [154,155]. The large hydrophobic terpene geranyl group covalently modifies the position 2 thio group of 2-thiouridine inducing a loss of protonation at the N3 atom of U 34 [156].…”