Thienoguanosine (^thG) is an isomorphic nucleoside analogue acting as a faithful fluorescent substitute of G, with respectable quantum yield in oligonucleotides. Photophysical analysis of ^thG reveals the existence of two ground-state tautomers with significantly shifted absorption and emission wavelengths, and high quantum yield in buffer. Using (TD)-DFT calculations, the tautomers were identified as the H1 and H3 keto-amino tautomers. When incorporated into the loop of (−)PBS, the (−)DNA copy of the HIV-1 primer binding site, both tautomers are observed and show differential sensitivity to protein binding. The red-shifted H1 tautomer is strongly favored in matched (−)/(+)PBS duplexes, while the relative emission of the H3 tautomer can be used to detect single nucleotide polymorphisms. These tautomers and their distinct environmental sensitivity provide unprecedented information channels for analyzing G residues in oligonucleotides and their complexes.

Thienoguanosine (^thG) is an isomorphic nucleoside analogue acting as a faithful fluorescent substitute of G, with respectable quantum yield in oligonucleotides. Photophysical analysis of ^thG reveals the existence of two ground-state tautomers with significantly shifted absorption and emission wavelengths, and high quantum yield in buffer. Using (TD)-DFT calculations, the tautomers were identified as the H1 and H3 keto-amino tautomers. When incorporated into the loop of (−)PBS, the (−)DNA copy of the HIV-1 primer binding site, both tautomers are observed and show differential sensitivity to protein binding. The red-shifted H1 tautomer is strongly favored in matched (−)/(+)PBS duplexes, while the relative emission of the H3 tautomer can be used to detect single nucleotide polymorphisms. These tautomers and their distinct environmental sensitivity provide unprecedented information channels for analyzing G residues in oligonucleotides and their complexes.

Nature's selection of the contemporary nucleobases in RNA and DNA continues to intrigue the origin of life community. While the prebiotic synthesis of the N-glycosyl bond has historically been a central area of investigation, variations in hydrolytic stability among the N-glycosyl bonds may have presented an additional selection pressure that contributed to nucleobase and nucleoside evolution. To experimentally probe this hypothesis, a systematic kinetic analysis of the hydrolytic deglycosylation reactions of modified, alternative, and native nucleosides was undertaken. Rate constants were measured as a function of temperature (at pH 1) to produce Arrhenius and Eyring plots for extrapolation to 37°C and determination of thermodynamic activation parameters. Rate enhancements based on the differences in reaction rates of deoxyriboglycosidic and riboglycosidic bonds were found to vary under the same conditions. Rate constants of deoxynucleosides were also measured across the pH range of 1–3 (at 50°C), which highlighted how simple changes to the heterocycle alone can lead to a significant variation in deglycosylation rates. The contemporary nucleosides exhibited the slowest deglycosylation rates in comparison with the nonnative/alternative nucleosides, which we suggest as experimental support for nature's selection of the fittest N-glycosyl bonds. Copyright © 2014 John Wiley & Sons, Ltd.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine (^thA). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that ^thA is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give ^thI. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the ^thA-modified RNA. The observed high ^thA reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine (^thA). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that ^thA is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give ^thI. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the ^thA-modified RNA. The observed high ^thA reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Guanidinoglycosides, a family of cellular transporters capable of delivering high M_w biopolymers, have previously been shown to display high selectivity for cell-surface heparan sulfate proteoglycans and promote their clustering. Herein, the internalization mechanism of amphiphilic guanidinoglycoside derivatives was investigated by cell-surface FRET analysis. Unexpectedly, although the heparan sulfate selectivity is maintained, the cellular uptake of these derivatives does not appear to involve clustering of the proteoglycans on the cell surface. This suggests a distinct uptake mechanism when compared to the parent guanidinoglycoside-based carriers.

Semisynthetic derivatives of the clinically useful aminoglycosides tobramycin and amikacin were prepared by selectively modifying their 6′′ positions with a variety of hydrogen bond donors and acceptors. Their binding to the rRNA A-site was probed using an in vitro FRET-based assay, and their antibacterial activities against several resistant strains (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, MRSA) were quantified by determining minimum inhibitory concentrations (MICs). The most potent derivatives were evaluated for their eukaryotic cytotoxicity. Most analogues displayed higher affinity for the bacterial A-site than the parent compounds. Although most tobramycin analogues exhibited no improvement in antibacterial activity, several amikacin analogues showed potent and broad-spectrum antibacterial activity against resistant bacteria. Derivatives tested for eukaryotic cytotoxicity exhibited minimal toxicity, similar to the parent compounds.

Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the 2P excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both thiophene-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications.

The native bases of RNA and DNA are prominent examples of the narrow selection of organic molecules upon which life is based. How did nature “decide” upon these specific heterocycles? Evidence suggests that many types of heterocycles could have been present on the early Earth. It is therefore likely that the contemporary composition of nucleobases is a result of multiple selection pressures that operated during early chemical and biological evolution. The persistence of the fittest heterocycles in the prebiotic environment towards, for example, hydrolytic and photochemical assaults, may have given some nucleobases a selective advantage for incorporation into the first informational polymers. The prebiotic formation of polymeric nucleic acids employing the native bases remains, however, a challenging problem to reconcile. Hypotheses have proposed that the emerging RNA world may have included many types of nucleobases. This is supported by the extensive utilization of non-canonical nucleobases in extant RNA and the resemblance of many of the modified bases to heterocycles generated in simulated prebiotic chemistry experiments. Selection pressures in the RNA world could have therefore narrowed the composition of the nucleic acid bases. Two such selection pressures may have been related to genetic fidelity and duplex stability. Considering these possible selection criteria, the native bases along with other related heterocycles seem to exhibit a certain level of fitness. We end by discussing the strength of the N-glycosidic bond as a potential fitness parameter in the early DNA world, which may have played a part in the refinement of the alphabetic bases.

Optimized facile syntheses and highly desirable spectroscopic properties of two isomorphic fluorescent pyrimidines, comprising a 1,2,4-triazine motif conjugated to a thiophene (1 a) or a furan (1 b), are described. Although structurally related to their 5-modified uridine counterparts, these modified 6-aza-uridines reveal dramatically improved fluorescence properties and a remarkable sensitivity to polarity and pH changes. The thiophene derivative 1 a has an absorption maximum around 335 nm, which upon excitation yields visible emission with a polarity-sensitive maximum and fluorescence quantum yield ranging from 415 nm (Φ=0.8) to 455 nm (Φ=0.2) in dioxane and water, respectively. Nucleoside 1 a also displays susceptibility to acidity. Correlating emission intensity and solution pH yields a pK_a value of 6.7–6.9, reasonably close to physiological pH values. The results illustrate that highly sought-after fluorescence features (brightness and responsiveness) are not necessarily the trait of large fluorophores alone, but can be observed with probes that meet stringent isomorphic design criteria.

The emergence of virulent, drug-resistant bacterial strains coupled with a minimal output of new pharmaceutical agents to combat them makes this a critical time for antibacterial research. Aminoglycosides are a well-studied, highly potent class of naturally occurring antibiotics with scaffolds amenable to modification, and therefore, they provide an excellent starting point for the development of semisynthetic, next-generation compounds. To explore the potential of this approach, we synthesized a small library of aminoglycoside derivatives selectively and minimally modified at one or two positions with a guanidine group replacing the corresponding amine or hydroxy functionality. Most guanidino-aminoglycosides showed increased affinity for the ribosomal decoding rRNA site, the cognate biological target of the natural products, when compared with their parent antibiotics, as measured by an in vitro fluorescence resonance energy transfer (FRET) A-site binding assay. Additionally, certain analogues showed improved minimum inhibitory concentration (MIC) values against resistant bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA). An amikacin derivative holds particular promise with activity greater than or equal to the parent antibiotic in the majority of bacterial strains tested.

Fluorescent nucleoside analogs, commonly used to explore nucleic acid dynamics, recognition and damage, frequently respond to a single environmental parameter. Herein we address the development of chromophores that can simultaneously probe more than one environmental factor while having each associated with a unique spectroscopic signature. We demonstrate that an isomorphic emissive pyridine-modified 2-deoxy-uridine 1, containing multiple sensory elements, responds to changes in acidity, viscosity, and polarity. Protonation of the pyridine moiety (pK_a 4.4) leads to enhanced emission (λ_em=388 nm) and red-shifted absorption spectra (λ_abs=319 nm), suggesting the formation of an intramolecular hydrogen bond with the neighboring pyrimidine carbonyl. This “locked” conformation can also be mimicked by increasing solvent viscosity, resulting in a stark enhancement of emission quantum yield. Finally, increasing solvent polarity substantially impacts the chromophore’s Stokes shift [from 5.8×10³ cm⁻¹ at E_T(30)=36.4 kcal mol⁻¹ to 9.3 ×10³ cm⁻¹ at E_T(30)=63.1 kcal mol⁻¹]. The opposite effect is seen for the impact of solvent polarity of the protonated form. The characteristic photophysical signature induced by each parameter facilitates the exploration of these environmental factors both individually and simultaneously.

Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin–phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.