Base pairing and structural insights into the 5-formylcytosine in RNA duplex.

5-Formylcytidine (f(5)C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m(5)C) through 5-hydroxymethylcytidine (hm(5)C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f(5)C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5'-GUA(f(5)C)GUAC-3']2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f(5)C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex.

Synthesis and investigation of the 5-formylcytidine modified, anticodon stem and loop of the human mitochondrial tRNAMet.

Human mitochondrial methionine transfer RNA (hmtRNA(Met)(CAU)) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f(5)C(34)). The role of this modification in (hmtRNA(Met)(CAU)) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

DNA polymerase bypass in vitro and in E. coli of a C-nucleotide analogue of Fapy-dG.

Bypass of the configurationally stable analogue (beta-C-Fapy x dG) of the formamidopyrimidine lesion derived from 2'-deoxyguanosine oxidation (Fapy x dG) was studied in vitro and in Escherichia coli. The exonuclease deficient Klenow fragment of E. coli DNA polymerase I (Klenow exo(-)) misincorporated dA most frequently opposite beta-C-Fapy x dG, but its efficiency was <0.2% of dC insertion. Klenow exo(-) fidelity was enhanced by the enzyme's high selectivity for extending duplexes only when dC was opposite beta-C-Fapy x dG. The expectations raised by these in vitro data were realized when beta-C-Fapy x dG replication was studied in E. coli by transfecting M13mp7(L2) bacteriophage DNA containing the nucleotide analogue within the lacZ gene in 4 local sequence contexts. The bypass efficiency of beta-C-Fapy x dG varied between 45% and 70% compared to a genome containing only native nucleotides. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay [Delaney, J. C.; Essigmann, J. M. Methods Enzymol.2006, 408, 1]. The levels of mutations could not be distinguished between those observed when genomes containing native nucleotides were replicated, indicating that the mutagenicity of beta-C-Fapy x dG was <1%. These data and previous reports indicate that beta-C-Fapy x dG is a good model of Fapy x dG in E. coli. In addition, these results and the previous report of beta-C-Fapy x dG binding to the base excision repair protein formamidopyrimidine glycosylase suggest that this analogue could be useful as a DNA repair inhibitor.

The human mitochondrial tRNAMet: structure/function relationship of a unique modification in the decoding of unconventional codons.

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA(Met)(CAU), to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA(Met)(CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f(5)C(34)), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL(Met)(CAU)), determined by NMR restrained molecular modeling, revealed how the f(5)C(34) modification facilitates the decoding of AUA at the P- and the A-sites. The f(5)C(34) defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL(Met)(CAU). The hmtASL(Met)(CAU) exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f(5)C(34) facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f(5)C(34) enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f(5)C(34), expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

Chromophoric 5'-O-Silyl protection of N-protected 2'-ACE ribonucleosides for solid-phase RNA synthesis.

Recent advances in the understanding of the pivotal roles played by endogenous small RNAs in gene regulation have resulted in a substantial and rapidly growing market for synthetic RNA. 5'-Silyl-2'-ACE chemistry has proven to be a robust and reliable technology for the synthesis of oligoribonucleotides. This unit describes an important improvement to this chemistry, by adding a cycle-to-cycle traceability analogous to that inherent in 5'-dimethoxytrityl-based approaches. This is achieved by first regioselectively introducing a 5'-alkynylsilyl protecting group onto the 2'-ACE-protected nucleosides. The 5'-alkynylsilyl group is then reacted with an azide derivative of the chromophore Disperse Red 1, which enables spectrophotometric interrogation of each coupling step following 5'-deprotection. Finally, the protected nucleosides are elaborated into their 3'-phosphoramidite derivatives for use in solid-phase RNA synthesis.

Synthesis of oligonucleotides and thermal stability of duplexes containing the beta-C-nucleoside analogue of Fapy*dG.

The formamidopyrimidine lesions (Fapy.dA, Fapy.dG) are formed in significant amounts when DNA is exposed to oxidative stress. These lesions are unusual in that they readily epimerize in solution. The distribution of configurational isomers in DNA is unknown. Nonepimerizable, nonhydrolyzable analogues are useful probes for investigating the configuration of Fapy lesions in DNA and as potential enzyme inhibitors. The beta-C-nucleoside of Fapy.dG has been prepared and introduced sight-specifically into oligonucleotides via its respective beta-cyanoethyl phosphoramidite. The phosphoramidite was prepared via a Wittig reaction between a protected form of deoxyribose and a suitably functionalized pyrimidine. The pyrimidine contained methyl and 2-propyl groups at the O4 and O2 positions, respectively, to differentiate between them following C-nucleoside formation. The formamide was derived from a nitro group at C5. The phosphoramidite coupled in 80% yield via a single 15-min coupling using tetrazole as activator. Oligonucleotides as long as 36 nucleotides were prepared and characterized by ESI-MS.

Interaction of DNA containing Fapy.dA or its C-nucleoside analogues with base excision repair enzymes. Implications for mutagenesis and enzyme inhibition.

Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.

Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV.

The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative stress. These lesions readily epimerize in water, an unusual property for nucleosides. The equilibrium mixture slightly favors the beta-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise alpha-deoxyadenosine was used as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA. Endo IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-anomer. Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate. Fapy.dA incision is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion. Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing alpha-Fapy.dA. The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-anomer in DNA. Endo IV incises Fapy.dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its beta-anomer in DNA.

Repair of DNA containing Fapy.dG and its beta-C-nucleoside analogue by formamidopyrimidine DNA glycosylase and MutY.

Fapy.dG is produced in DNA as a result of oxidative stress. Under some conditions Fapy.dG is formed in greater yields than 8-oxodG from a common chemical precursor. Recently, Fapy.dG and its C-nucleoside analogue were incorporated in chemically synthesized oligonucleotides at defined sites. Like 8-oxodG, Fapy.dG instructs DNA polymerase to misincorporate dA opposite it in vitro. The interactions of DNA containing Fapy.dG or the nonhydrolyzable analogue with Fpg and MutY are described. Fpg excises Fapy.dG (K(M) = 2.0 nM, k(cat) = 0.14 min(-1)) opposite dC approximately 17-fold more efficiently than when mispaired with dA, which is misinserted by DNA polymerase in vitro. Fpg also prefers to bind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in which the lesion is opposite dA. MutY incises dA when it is opposite Fapy.dG and strongly binds duplexes containing the lesion or beta-C-Fapy.dG. Incision from Fapy.dG.dA is faster than from dG.dA mispairs but slower than from DNA containing 8-oxodG opposite dA. These data demonstrate that Fapy.dG closely resembles the interactions of 8-oxodG with two members of the GO repair pathway in vitro. The similar effects of Fapy.dG and 8-oxodG on DNA polymerase and repair enzymes in vitro raise the question as to whether Fapy.dG elicits similar effects in vivo.

Synthesis and characterization of oligodeoxynucleotides containing formamidopyrimidine lesions and nonhydrolyzable analogues.

Oligodeoxynucleotides containing formamidopyrimidine lesions and C-nucleoside analogues at defined sites were prepared by solid-phase synthesis and in some cases enzymatic ligation. Formamidopyrimidine lesions were introduced as dinucleotides to prevent rearrangement to their pyranose isomers. Oligodeoxynucleotides containing single diastereomers of C-nucleoside analogues of Fapy.dA were introduced by using the respective phosphoramidites. The formamidopyrimidine lesions reduce the T(M) of dodecamers relative to their unmodified nucleotide counterparts when opposite the nucleotide proper base-pairing partner. However, duplexes containing Fapy.dG-dA mispairs melt significantly higher than those comprised of dG-dA. All duplexes containing Fapy.dA-dX or its C-nucleoside analogue melt lower than the respective complexes containing dA-dX. Studies of the alkaline lability of oligodeoxynucleotides containing formamidopyrimidine lesions indicate that Fapy.dA is readily identified as an alkali-labile lesion with use of piperidine (1.0 M, 90 degrees C, 20 min), but Fapy.dG is less easily identified in this manner.