Synthesis of RNA Containing 5-Hydroxymethyl-, 5-Formyl-, and 5-Carboxycytidine.

5-Hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. The same bases (5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine; hmC, fC, and caC) have now also been detected in mammalian RNA with a high abundance in mRNA. While DNA phosphoramidites (PAs) that allow the synthesis of xdC-containing oligonucleotides for deeper biological studies are available, the corresponding silyl-protected RNA PAs for fC and caC have not yet been disclosed. Here, we report novel RNA PAs for hmC, fC, and caC that can be used in routine RNA synthesis. The new building blocks are compatible with the canonical PAs and also with themselves, which enables even the synthesis of RNA strands containing all three of these bases. The study will pave the way for detailed physical, biochemical, and biological studies to unravel the function of these non-canonical modifications in RNA.

Fluorescence enhancement of oligodeoxynucleotides modified with green fluorescent protein chromophore mimics upon triplex formation.

Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides. These oligonucleotides were designed to form triplex DNA in order to encapsulate the GFP chromophores, mimicking GFP structures. Upon triplex formation, the embedded GFP chromophores exhibited fluorescence enhancement, suggesting the potential application of these fluorescent probes for the detection of nucleic acids.

Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.

Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition.

We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate.

Enzymatic synthesis and the reverse transcription of RNAs containing 2'-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2'-O-carbamoyl uridine triphosphate (UcmTP). UcmTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection.

Synthesis of 5'-terminal capped oligonucleotides using O-N phosphoryl migration of phosphoramidite derivatives.

Trivalent phosphoramidite derivatives could be readily converted by reacting with 1-hydroxy-7-azabenzotriazole to phosphotriester intermediates; these intermediates reacted smoothly with phosphorylated compounds to give pyrophosphate derivatives. This new phosphorylation approach enabled a facile and rapid synthesis of 5'-adenylated DNA oligomers (A(5')ppDNA) on resins using a silyl-type linker. Our new approach could be applied to the synthesis of a 2'-OMe-RNA oligomer containing the 5'-terminal 2,2,7-trimethylguanosine cap structure.