Synthesis of 5-Fluoropyrimidine Nucleotides as Sensitive NMR Probes of RNA Structure

Abstract

Enzymatic synthesis methods for the fluorinated 5'-triphosphate analogues 5F-UTP and 5F-CTP have been developed to facilitate 19F-labeling of RNAs for biophysical studies. HIV-2 TAR RNAs were synthesized using these analogues by in vitro transcription reactions using T7 RNA polymerase. The uniform incorporation of 5F-U or 5F-C analogues into HIV-2 TAR RNA transcripts does not significantly alter the RNA structure or thermodynamic stability. Fluorine observed homonuclear 19F-19F and heteronuclear 19F-1H NOE experiments providing selective distance information are presented and discussed. The availability of efficient synthesis of 5F-UTP, and for the first time, 5F-CTP, will facilitate the use of 5F-labeled RNAs in structural, ligand binding, and dynamic studies of RNAs using the advantages of 19F-labeling.

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

“…We then exploited different chemical shifts of 5-19 F-incorporated pyrimidine nucleotide, in either a single-or doublestranded region of RNA, which has previously been used to probe the secondary structure of RNA (Horowitz et al 1977;Marshall and Smith 1977;Gollnick et al 1986;Chu et al 1992;Kanyo et al 1996;Sahasrabudhe and Gmeiner 1997;Arnold and Fisher 2000;Hammann et al 2001;Olejniczak et al 2002;Hennig et al 2007;Puffer et al 2009) to demonstrate the presence of an alternative conformation of U2-U6 snRNA complex conclusively. The advantage of observing the 19 F nucleus by NMR is the broader range and the greater sensitivity of fluorine chemical shifts in response to the local environment as compared to those of hydrogen because the fluorine nucleus is surrounded by nine electrons vs. a single electron in hydrogen.…”

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

“…We, and others, have demonstrated that the introduction of 19 F substitutions into the heterocyclic bases is nonperturbing and provides uniquely positioned, sensitive NMR reporter groups to monitor conformational properties of modified RNA (Hennig, Scott, Sperling, Bermel, & Williamson, 2007;Puffer et al, 2009;Scott, Geierstanger, Williamson, & Hennig, 2004). Additional advantages of using spin I ¼ ½ 19 F as an NMR probe are its high sensitivity (83% of the 1 H) combined with 100% natural abundance and a wide chemical shift distribution ($50-fold larger than that of 1 H) (Kitevski-LeBlanc & Prosser, 2012;Rastinejad et al, 1995).…”

“…Here, we describe a combination of RNA-labeling techniques as well as NMR methods and applications to a well-established model system, the human immunodeficiency virus (HIV) transactivation response element (TAR) (Fischer et al, 1996;Fischer, Gdaniec, Olejniczak, Bielecki, & Adamiak, 1999;Hennig et al, 2006Hennig et al, , 2007Huang, Varani, & Drobny, 2010; Olejniczak et al, 2002;Olsen et al, 2005;Scott et al, 2004). The Tat (transcription activator)-TAR complex formation provides an essential gene regulatory function for HIV (Marciniak, Calnan, Frankel, & Sharp, 1990;Seelamgari et al, 2004).…”

“…We established efficient and economical enzymatic syntheses for the fluorinated nucleotide analogues 5-fluorouridine-5 0 -triphosphate (5F-UTP), 5-fluorocytidine-5 0 -triphoshate (5F-CTP) (Hennig et al, 2006(Hennig et al, , 2007, and 2-fluoroadenosine-5 0 -triphosphate (2F-ATP) (Scott et al, 2004) and demonstrated that the nucleotides can be specifically incorporated into RNA using standard DNA template-directed T7 RNA polymerase methods (Fig. 3).…”

A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

“…The preparation of 5-hydroxycytidine (oh 5 C) was carried out using an adapted in vitro enzymatic synthesis method for 5-fluorouridine-5′-triphosphate [31]. The synthesis of oh 5 C began with the C-5-phosphorylation of ribose by ribokinase (rbsK), to give ribose-5-phosphate (R5P) that is further phosphorylated at the C-1 position by 5-phospho-D-ribosyl-α-1-pyrophosphate synthase (prsA) forming 5-phospho-Dribosyl-α-1-pyrophosphate (PRPP).…”