7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

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 A→T 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.

Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring.

The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM-BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands.

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues.

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.

Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges.

We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.

Benzothiazole-based cyanines as fluorescent "light-up" probes for duplex and quadruplex DNA.

Analogs of benzothiazole orange (BO) with one, two or three methylbenzothiazolylmethylidene substituents in the 1-methylpyridinium ring were obtained from the respective picolinium, lutidinium or collidinium salts. Fluorescence parameters of the known and new dyes in complexes with various DNA structures, including G-quadruplexes (G4s) and i-motifs (IMs), were analyzed. All dyes efficiently distinguished G4s and ss-DNA. The bi- and tri-substituted derivatives had basically similar distributions of relative fluorescence intensities. The mono-substituted derivatives exhibited enhanced sensitivity to parallel G4s. All dyes were particularly sensitive to a G4 structure with an additional duplex module (the thrombin-binding aptamer TBA31), presumably due to a distinctive binding mode (interaction with the junction between the two modules). In particular, BO showed a strong (160-fold) enhancement in fluorescence quantum yield in complex with TBA31 compared to the free dye. The fluorescence quantum yields of the 2,4-bisubstituted derivative in complex with well-characterized G4s from oncogene promoters were in the range of 0.04-0.28, i.e. comparable to those of ThT. The mono/bi-substituted derivatives should be considered as possible light-up probes for G4 formation.

DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors.

I-motif structures, adopted by cytosine-rich DNA strands, have attracted considerable interest as possible regulatory elements in genomes. Applied science exploits the advantages of i-motif stabilization under acidic conditions: i-motif-based pH sensors and other biocompatible nanodevices are being developed. Two key characteristics of i-motifs as core elements of nanodevices, i.e., their stability under physiological conditions and folding/unfolding rates, still need to be improved. We have previously reported a phenoxazine derivative (i-clamp) that enhances the thermal stability of the i-motif and shifts the pH transition point closer to physiological values. Here, we performed i-clamp guanidinylation to further explore the prospects of clamp-like modifications in i-motif fine-tuning. Based on molecular modeling data, we concluded that clamp guanidinylation facilitated interstrand interactions in an i-motif core and ultimately stabilized the i-motif structure. We tested the effects of guanidino-i-clamp insertions on the thermal stabilities of genomic and model i-motifs. We also investigated the folding/unfolding kinetics of native and modified i-motifs under moderate, physiologically relevant pH alterations. We demonstrated fast folding/unfolding of native genomic and model i-motifs in response to pH stimuli. This finding supports the concept of i-motifs as possible genomic regulatory elements and encourages the future design of rapid-response pH probes based on such structures. Incorporation of guanidino-i-clamp residues at/near the 5'-terminus of i-motifs dramatically decreased the apparent unfolding rates and increased the thermal stabilities of the structures. This counterplay between the effects of modifications on i-motif stability and their effects on kinetics should be taken into account in the design of pH sensors.