Nevertheless, ethidium bromide is well-known for its toxicity and mutagenic properties 7. Ethidium bromide is a classic example of a DNA intercalator that became highly fluorescent upon binding to double-stranded DNA (dsDNA) thus it used to be a popular staining agent for visualizing nucleic acids following gel electrophoresis 4, 5, 6. These dyes are typically conjugated organic molecules that can bind non-covalently to nucleic acids in one or more of several possible modes including intercalation, groove binding, aggregation, and electrostatic interaction 3. In recent years, fluorescent nucleic acid stains have become an essential tool for qualitative and quantitative analysis of nucleic acids both in vitro and in the cells. Alternatively, the use of smart labels or probes that can exhibit the signal in response to the nucleic acid structures or sequences of interest allows the assay to be performed in a homogeneous format that is more convenient and can be applied for the detection of nucleic acid targets in living cells. This is typically performed in a heterogeneous assay format whereby the bound and unbound labels (or labeled probes) are partitioned in separated phases, followed by washing to eliminate the unbound labels or probes. For a label that exhibits permanent signals, a mechanism to distinguish between positive and negative events must be incorporated. The latter is more convenient as no complicated modification and purification steps are required although the specificity may not be as good as the probe-based assay. The label may be attached to the nucleic acid molecule either through a covalent bond 1 to form a probe, or by noncovalent binding to a specific sequence or structure of nucleic acid targets 2. However, since nucleic acid molecules themselves have no intrinsic properties that allow direct optical detection with sufficient sensitivity, a signaling label is often required to improve the sensitivity of the assays. Optical detection of nucleic acids is important because nucleic acids carry genetic information and play indispensable roles in the production of proteins that are essential for the structure and functions of all organisms. Applications of the developed dyes for colorimetric detection of DNA in vitro and imaging of cellular nucleic acids are also demonstrated. The binding interaction and optical response of the dicationic styryl dyes with nucleic acid were superior to the corresponding monocationic styryl dyes.

Optical properties of the newly synthesized styryl dyes have been studied in the presence and absence of nucleic acid targets with the aim to find new dyes that can sensitively and specifically change fluorescence and/or color in the presence of nucleic acid targets. In this work, novel dicationic styryl dyes bearing quaternary ammonium groups are designed to improve binding strength and optical response with target nucleic acids which contain a negatively charged phosphate backbone. In addition to fluorescence, styryl dyes are strongly colored and exhibit solvatochromic properties which make them useful as colorimetric stains for low-cost and rapid testing of nucleic acids. Styryl dyes have recently gained popularity as potential biological staining agents with many appealing properties, including a straightforward synthesis procedure, excellent photostability, tunable fluorescence, and high fluorescence quantum yield in the presence of nucleic acid targets with low background fluorescence signals. As a result, finding nontoxic, easily accessible dyes, with desirable optical characteristics remains important. Nevertheless, the range of commercially accessible dyes is still rather limited, and they are often very costly. Nucleic acid staining dyes are important tools for the analysis and visualizing of DNA/RNA in vitro and in the cells.

Groove agent 4.2