Isomorphic Emissive GTP Surrogate Facilitates Initiation and Elongation of in Vitro Transcription Reactions

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 ^thGTP, a newly synthesized isomorphic fluorescent nucleotide with a thieno­[3,4-<i>d</i>]­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 ^thG 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 ^thG 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 ^thG 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

Chemical Mutagenesis of an Emissive RNA Alphabet

An evolved fluorescent ribonucleoside alphabet comprising isomorphic purine (^<b>tz</b><b>A</b>, ^<b>tz</b><b>G</b>) and pyrimidine (^<b>tz</b><b>U</b>, ^<b>tz</b><b>C</b>) analogues, all derived from isothiazolo­[4,3-<i>d</i>]­pyrimidine as a common heterocyclic core, is described. Structural and biochemical analyses illustrate that the nucleosides, particularly the <i>C</i>-nucleosidic purine analogues, are faithful isomorphic and isofunctional surrogates of their natural counterparts and show improved features when compared to an RNA alphabet derived from thieno­[3,4-<i>d</i>]-pyrimidine. The restoration of the nitrogen in a position equivalent to the purines’ N7 leads to “isofunctional” behavior, as illustrated by the ability of adenosine deaminase to deaminate ^<b>tz</b><b>A</b> as effectively as adenosine, the native substrate

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD⁺ Analogue

The synthesis, photophysics, and biochemical utility of a fluorescent NAD⁺ analogue based on an isothiazolo­[4,3-<i>d</i>]­pyrimidine core (<b>N</b>^<b>tz</b><b>AD</b>^<b>+</b>) are described. Enzymatic reactions, photophysically monitored in real time, show <b>N</b>^<b>tz</b><b>AD</b>^<b>+</b> and <b>N</b>^<b>tz</b><b>ADH</b> to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as <b>N</b>^<b>tz</b><b>AD</b>^<b>+</b> is converted to <b>N</b>^<b>tz</b><b>ADH</b>, reflecting a complementary photophysical behavior to that of the native NAD⁺/NADH. <b>N</b>^<b>tz</b><b>AD</b>^<b>+</b> and <b>N</b>^<b>tz</b><b>ADH</b> serve as substrates for NADase, which selectively cleaves the nicotinamide’s glycosidic bond yielding ^<b>tz</b><b>ADP-ribose</b>. <b>N</b>^<b>tz</b><b>AD</b>^<b>+</b> also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer ^<b>tz</b><b>ADP-ribose</b> to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD⁺

GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery

GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle

Design, Synthesis, and Spectroscopic Properties of Extended and Fused Pyrrolo-dC and Pyrrolo-C Analogs

The syntheses of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a thiophene moiety, are described. A thorough photophysical analysis of the nucleosides, in comparison to PyC, is reported

Visibly Emissive and Responsive Extended 6‑Aza-Uridines

A family of extended 5-modified-6-aza-uridines was obtained via Suzuki coupling reactions with a common brominated precursor. Extending the conjugated-6-aza-uridines with substituted aryl rings increases the push–pull interactions yielding enhanced bathochromic shifts and solvatochromism compared to the parent nucleosides. For example, the methoxy substituted derivative <b>1d</b> displays λ_max abs around 375 nm, with visible emission maxima at 486 nm (Φ = 0.74) and 525 nm (Φ = 0.02) in dioxane and water, respectively

Oligodeoxynucleotides Containing Multiple Thiophene-Modified Isomorphic Fluorescent Nucleosides

5-(Thien-2-yl)-2′-deoxyuridine, an isomorphic fluorescent nucleoside analogue, was incorporated into multiple positions within single stranded oligodeoxynucleotides. With minimal impact on duplex stability and overall structure, oligonucleotides containing three identical isomorphic fluorescent nucleosides in alternating or neighboring positions display enhanced, sequence-dependent on-signals for either duplex formation or dissociation

Aggregation-Mediated Macromolecular Uptake by a Molecular Transporter

Endocytosis is a key process in cellular delivery of macromolecules by molecular transporters, although the mechanism of internalization remains unclear. Here, we probe the cellular uptake of streptavidin using biotinylated guanidinoneomycin (biotinGNeo), a low molecular weight guanidinium-rich molecular transporter. Two distinct modes were explored: (i) incubation of cells with a preformed tetravalent streptavidin-(biotinGNeo)₄ conjugate and (ii) preincubation of cells with the biotinGNeo before exposure to streptavidin. A significant enhancement in uptake was observed after preincubation with biotinGNeo. FRET studies showed that the enhanced uptake was accompanied by extensive aggregation of streptavidin on the cell surface. Because guanidinylated neomycin was previously found to exclusively bind to heparan sulfate, our observations suggest that heparan sulfate proteoglycan aggregation is a pivotal step for endocytic entry into cells by guanidinoglycosides. These observations put forward a practical and general pathway for the cellular delivery of diverse macromolecules

Direct Observation of Aminoglycoside–RNA Binding by Localized Surface Plasmon Resonance Spectroscopy

RNA is involved in fundamental biological functions when bacterial pathogens replicate. Identifying and studying small molecules that can interact with bacterial RNA and interrupt cellular activities is a promising path for drug design. Aminoglycoside (AMG) antibiotics, prominent natural products that recognize RNA specifically, exert their biological functions by binding to prokaryotic ribosomal RNA and interfering with protein translation, ultimately resulting in bacterial cell death. The decoding site, a small internal loop within the 16S rRNA, is the molecular target for the AMG antibiotics. The specificity of neomycin B, a highly potent AMG antibiotic, to the ribosomal decoding RNA site, was previously studied by observing AMG–RNA complexes in solution. Here, we study this interaction using localized surface plasmon resonance (LSPR) transducers comprising gold island films prepared by evaporation on glass and annealing. Small molecule AMG receptors were immobilized on the Au islands via polyethylene glycol (PEG)-thiol linkers, and the interaction with target RNA in solution was studied by monitoring the change in the LSPR optical response upon binding. The results show high-affinity binding of neomycin to 27-nucleotide model A-site RNA sequence in the nanomolar range, while no specific binding is observed for synthetic RNA oligomers (e.g., poly-U). The impact of specific base substitutions in the A-site RNA constructs on binding affinity and selectivity is determined quantitatively. It is concluded that LSPR is a powerful tool for providing molecular insight into small molecule–RNA interactions and for the design and screening of selective antimicrobial drugs

Additional file 1: of Surfen and oxalyl surfen decrease tau hyperphosphorylation and mitigate neuron deficits in vivo in a zebrafish model of tauopathy

Figure S1. Percentage of embryonic survival observed for 72 hpf wild-type (WT) and Tg[HuC::hTauP301L; DsRed] (non-treated) embryos incubated for 2 days in E3 medium containing 1% DMSO or E3 medium containing 1% DMSO with LiCl (10–150 mM) (a), surfen (0.1–10 μM) (b), oxalyl surfen (0.1–10 μM) (c) or hemisurfen (0.1–10 μM) (d). Note that at the selected concentrations (80 mM LiCl, 3 μM for surfen and hemisurfen and 2 μM for oxalyl surfen) are the maximal non-toxic concentrations (n = 250, *P < 0.05, ***P < 0.001, ns: non-significant, Student’s t test). (TIFF 55462 kb