The fastidious behavior of T7 RNA polymerase limits the incorporation of synthetic nucleosides into RNA transcripts, particularly at or near the promoter. The practically exclusive use of GTP for transcription initiation further compounds this challenge, and reactions with GTP analogs, where the heterocyclic nucleus has been altered, have not, to our knowledge, been demonstrated. The enzymatic incorporation of (th)GTP, a newly synthesized isomorphic fluorescent nucleotide with a thieno[3,4-d]pyrimidine core, is explored. The modified nucleotide can initiate and maintain transcription reactions, leading to the formation of fully modified and highly emissive RNA transcripts with (th)G replacing all guanosine residues. Short and long modified transcripts are synthesized in comparable yields to their natural counterparts. To assess proper folding and function, transcripts were used to assemble a hammerhead ribozyme with all permutations of natural and modified enzyme and substrate strands. The (th)G modified substrate was effectively cleaved by the natural RNA enzyme, demonstrating the isomorphic features of the nucleoside and its ability to replace G residues while retaining proper folding. In contrast, the (th)G modified enzyme showed little cleavage ability, suggesting the modifications likely disrupted the catalytic center, illustrating the significance of the Hoogsteen face in mediating appropriate contacts. Importantly, the ribozyme cleavage reaction of the emissive fluorescent transcripts could be followed in real time by fluorescence spectroscopy. Beyond their utility as fluorescent probes in biophysical and discovery assays, the results reported point to the potential utility of such isomorphic nucleosides in probing specific mechanistic questions in RNA catalysis and RNA structural analysis.

To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.

Adenosine deaminase, a major enzyme involved in purine metabolism, converts an isomorphic fluorescent analogue of adenosine (thA) to an isomorphic inosine analogue (thI), which possesses distinct spectral features, allowing one to monitor the enzyme-catalyzed reaction and its inhibition in real time. The utility of this sensitive fluorescently-monitored transformation for the high throughput detection and analysis of ADA inhibitors is demonstrated.

Polymyxin B is an antibiotic used against multi-resistant gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating toxin. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.

Polymyxin B is an antibiotic used against multi-resistant gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating toxin. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.

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.

Looking for targets: while the bactericidal activity of polymyxins is attributed to changes in membrane permeation, we show that these antibiotics can bind prokaryotic and eukaryotic A-sites, domains responsible for translational decoding. Polymyxin B, colistin and analogues also hinder eukaryotic translation in vitro. These new targets and effects might be partially responsible for the plethora of adverse effects by these potent bactericidal agents.