RESUMEN
Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.
Asunto(s)
Colorantes Fluorescentes , Macrófagos , Mangifera , ARN Pequeño no Traducido , Aptámeros de Nucleótidos/genética , Fluorescencia , Colorantes Fluorescentes/metabolismo , Mangifera/genética , Mangifera/metabolismo , ARN/metabolismo , Macrófagos/microbiologíaRESUMEN
This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly AâT transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.
Asunto(s)
Escherichia coli , Timina , Emparejamiento Base , ADN/genética , Reparación del ADN , Escherichia coli/genéticaRESUMEN
Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.
Asunto(s)
Colorantes Fluorescentes/química , G-Cuádruplex , Proteínas Fluorescentes Verdes/química , Fluorescencia , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligonucleótidos/química , Temperatura de TransiciónRESUMEN
Rose Bengal (RB) is an anionic xanthene dye with multiple useful biological features, including photosensitization properties. RB was studied extensively as a photosensitizer, mostly for antibacterial and antitumor photodynamic therapy (PDT). The application of RB to virus inactivation is rather understudied, and no RB derivatives have been developed as antivirals. In this work, we used a synthetic approach based on a successful design of photosensitizing antivirals to produce RB derivatives for virus photoinactivation. A series of n-alkyl-substituted RB derivatives was synthesized and evaluated as antiviral photosensitizers. The compounds exhibited similar 1O2 generation rate and efficiency, but drastically different activities against SARS-CoV-2, CHIKV, and HIV; with comparable cytotoxicity for different cell lines. Submicromolar-to-subnanomolar activities and high selectivity indices were detected for compounds with C4-6 alkyl (SARS-CoV-2) and C6-8 alkyl (CHIKV) chains. Spectrophotometric assessment demonstrates low aqueous solubility for C8-10 congeners and a significant aggregation tendency for the C12 derivative, possibly influencing its antiviral efficacy. Initial evaluation of the synthesized compounds makes them promising for further study as viral inactivators for vaccine preparations.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Rosa Bengala , Humanos , Rosa Bengala/farmacología , Rosa Bengala/química , SARS-CoV-2 , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Antivirales/farmacologíaRESUMEN
G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.