The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease mechanisms involving NAs.

Unravelling unique molecular targets specific to viruses is challenging yet critical for diagnosing emerging viral diseases. Nucleic acids and proteins are the major targets in diagnostic assays of viral pathogens. Identification of novel sequences and conformations of nucleic acids as targets is desirable for developing diagnostic assays specific to a virus of interest. Here, we disclose the identification and characterization of a highly conserved antiparallel G-quadruplex (GQ)-forming DNA sequence present within the SARS-CoV-2 genome. The two-quartet GQ with unique loop compositions formed a distinct recognition motif. Design, synthesis, and fine tuning of structure−activity of a set of small molecules led to the identification of a benzobisthiazole-based fluorogenic probe which unambiguously recognizes the target SARS-CoV-2 GQ DNA. A robust cost-effective assay was developed through thermal cycler PCR-based amplification of the antiparallel GQ-forming ORF1ab region of the SARS-CoV-2 genome and endpoint fluorescence detection with the probe. An exclusive pH window (3.5−4) helped trigger reliable conformational polymorphism (RCP) involving DNA duplex to GQ transformation, which aided the development of a GQ-RCP platform for the diagnosis of SARS-CoV-2 clinical samples. This general strategy can be adapted for the development of specific diagnostic assays targeting different noncanonical nucleic acid sequences.

Nucleic acid-based architectures have opened up numerous opportunities for basic and applied research in the field of DNA nanotechnology. The scheme of molecular architectonics of nucleic acids exploits conventional and unconventional base pairing interactions to integrate molecular partners in constructing functional molecular architectures and devices. The pH-responsive functional nucleic acid systems and devices have gained interest in diagnostics and therapeutics because of their biocompatibility and structural programmability. In this Mini-Review, we discuss recent advancements in the area of nucleic acid architectonics with a special emphasis on pH-driven molecular systems including molecular and nanoarchitectures, templated architectures and nanoclusters, nanomachines, hydrogels, targeted bioimaging, and drug delivery architectures. Finally, the Mini-Review is concluded by highlighting the challenges and opportunities for future developments.

The interplay of condensation and decondensation of DNA plays a crucial role in chromosome maintenance and gene expression. The molecular architectonics governing the chromatin condensation–decondensation cycle are worth studying, as DNA performs unique and distinct roles in each state and switches between two states without the loss of structural and functional integrity. This phenomenon has been adapted and implemented in transfection studies. Effective gene delivery into the cells to achieve respectable transfection efficiency has remained a challenge and emphasizes the need for understanding the steps involved in DNA delivery and transfection. Especially, recognizing the factors that effectively regulate DNA decondensation can provide logical solutions to the hurdles affecting the transfection efficiency. We designed a set of small molecule-based threading intercalation ligands as model condensing agents to study various factors influencing the DNA condensation and decondensation process. This study revealed condensation of DNA into nanocondensate by the threading intercalator and endogenous stimuli induced effective decondensation. Further, DNA nanocondensates are tracked using the intrinsic fluorescence in the lower pH of endocytic pathway and were evaluated as nonviral vectors for in cellulo delivery of plasmids. The correlation of decondensation of DNA nanocondensate with endogenous metal ions at their physiological concentrations provided valuable insights and implications for intracellular DNA delivery.

Pathogenic genomes harboring noncanonical G-quadruplex (GQ) forming sequences are potential targets for diagnosis. The GQ-forming cDNA sequences of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) are identified and validated as reliable...