A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification — the epitranscriptome — is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5′-iodide and a 3′-phosphorothioate. The sequence is amplified through rolling circle amplification with ϕ29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ∼50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ∼15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

N6-methyladenosine (m6A) is the most abundant mRNA modification, and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m6A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m6A. We show that the enzyme can be used to locate and quantify m6A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m6A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m6A in mammalian 28S rRNA is nucleotide 4190.

Formaldehyde is universally employed to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, and avoiding high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5–2.4 fold using a catalyst under optimized conditions, and by 7–25 fold compared to a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 10 3 -to 10 4 -fold, corresponding to free energy differences of ΔΔG inc ∼ 5.5-7 kcal/mol? Standard ΔΔG°values (∼0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper [Olson et al. (2013) J Am Chem Soc 135:1205-1208] used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent ΔG°= −RT ln K eq , indicating ΔΔG°of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore, ΔΔG°i nc values obtained from such steady-state evaluations of K eq are not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site. -fold, corresponding to free energy differences ΔΔG inc ∼ 5.5-7 kcal/mol (1). Kinetic studies have identified a variety of "checkpoints" favoring the selection of R over W. Kinetic checkpoints are triggered by substrate binding; the ternary pol-DNA-dNTP complex is stabilized with dRTP and destabilized with dWTP. The associated conformational changes drive the reaction forward toward incorporation with dRTP and backward toward substrate release with dWTP favoring incorporation of R over W (reviewed in refs. 1-4). The rate-determining steps can be different for different pols and may also differ for R and W for a single pol (3,4).A fundamental issue is to identify possible sources of free energy that might be large enough to account for high pol incorporation fidelity. Seemingly an obvious source might be the differences in stability of matched and mismatched base pairs in the DNA itself, which involve both H-bonding and base-stacking interactions (5). In an early experiment, used here as an example, equilibrium constants (K eq ) for double-stranded DNA (dsDNA) containing R and W base pairs at blunt-end termini were obtained by measuring melting temperatures in aqueous solution and used to infer standard free energy differences ΔΔG°∼ 0.3 kcal/mol (6). Because these were far too small to account for fidelity, it was then proposed that steric constraints imposed by the pol active site could "amplify" the small free energy differences between R and W base pairing to attain ΔΔG°...

We report a new strategy to produce luminescence signals from DNA synthesis by designing chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. We describe the synthesis of ATP-releasing nucleotides (ARNs) as derivatives of the four canonical nucleotides. We find that the four are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs are found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. Using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

Sealed tube microwaVe dielectric heating of diaryl acetylenes with cyclopentadienyl cobalt dicarbonyl at eleVated temperature in p-xylene proVides access to metallocenes in both the cyclobutadiene (Ar 4 C 4 CoCp) and cyclopentadienone (Ar 4 C 4 (CdO)CoCp) families. When compared with the traditional thermal approach, the current method offers dramatically reduced reaction times and, especially with respect to cyclopentadienone complexes, increased yields. In the case of an especially bulky diarylacetylene the microwaVe approach allows access to a complex that cannot be readily obtained under traditional thermal conditions. An initial microwaVe-promoted Sonogashira coupling may be employed for in situ generation of the diarylacetylene, although lower yields of the metallocene complexes are ultimately obtained.

Fluorescence labeling of DNAs is broadly useful, but methods for labeling are expensive and labor-intensive. Here we describe a general method for fluorescence labeling of oligonucleotides readily and cost-efficiently via base excision trapping (BETr), employing deaminated DNA bases to mark label positions, which are excised by base excision repair enzymes generating AP sites. Specially designed aminooxy-substituted rotor dyes trap the AP sites, yielding high emission intensities. BETr is orthogonal to DNA synthesis by polymerases, enabling multi-uracil incorporation into an amplicon and in situ BETr labeling without washing. BETr also enables labeling of dsDNA such as genomic DNA at a high labeling density in a single tube by use of nick translation. Use of two different deaminated bases facilitates two-color site-specific labeling. Use of a multi-labeled DNA construct as a bright fluorescence tag is demonstrated through the conjugation to an antibody for imaging proteins. Finally, double-strand selectivity of a repair enzyme is harnessed in sensitive reporting on the presence of a target DNA or RNA in a mixture with isothermal turnover and single nucleotide specificity. Overall, the results document a convenient and versatile method for general fluorescence labeling of DNAs.