RSC Adv.2016 6(90):87400-87404.

Selective recognition of G-quadruplexes by a dimeric carbocyanine dye

P. Chilka1, P. R. Patlolla2 and B. Datta1,2*

1Department of Biological Engineering, 2Department of Chemistry, Indian Institute of Technology, Gandhinagar, Gujarat, India

* Corresponding author

* Bhaskar Datta, Department of Chemistry & Department of Biological Engineering, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar 382355 India.

Telephone: 079-2395-2073; Fax: 079-2397-2622; E-mail: bdatta@iitgn.ac.in

 

Abstract :

Over the past two decades G-quadruplexes have emerged in pivotal roles across a plethora of physiological processes. The prevalence of putative G-quadruplex forming sequences in the human genome as well as a deeper understanding of the nuances underlying quadruplex structure have enabled researchers to explore these nucleic acid structures from diagnostic and therapeutic perspectives. The search for smart ligands that are capable of selective identification of G-quadruplexes compared to canonical duplex DNA have led to several promising leads to be identified. Ligands that are capable of selectively disrupting or modifying quadruplexes carry immense therapeutic potential. Quadruplex-specific ligands are expected to report the formation of such structures thereby enabling disease prognosis. Considering the polymorphism of quadruplexes, quadruplex-selective ligands can contribute towards deeper understanding of the dynamics of quadruplex topologies. They have also opened up the prospect of direct visualization of the structures in cells. The live cell imaging of quadruplex structures is challenging owing to the abundance of duplex DNA and other nucleic acids that can interfere with the probing agent. An ideal G-quadruplex specific fluorescent probe would possess attractive photostability, quantum yield of fluorescence, suitable cell permeability and low toxicity. We have designed a dimeric carbocyanine dye that displays high selectivity for certain G-quadruplex topologies in contrast to other nucleic acid secondary structures. Our dimeric carbocyanine dye is cell permeable and photostable. Our dye enables direct visualization of G-quadruplex structures in untreated live cells. Cellular imaging data reveals ability of the dye to stain nuclear regions and mitochondrial DNA. In particular, the brightly fluorescent spots in the nucleus likely correspond to guanine-rich rDNA involved in formation of temporal quadruplex conformations. The distinct fluorescent foci with low cytoplasmic fluorescence are promising for use of the dye for tracking quadruplex structure in vivo.

Key words: Live cell imaging, G-Quadruplexes, Fluorescence Probe

 

Supplement 

Cyanine dyes have been long examined for their interaction with nucleic acids as well as with other biomolecules. Cyanine dyes that are capable of self-assembly into fluorescence quenched aggregates can serve as an important tool in fluorescence turn-on assays upon encountering suitable target molecules. In this regard, we have prepared dimeric carbocyanine dyes that spontaneously self-aggregate into H-dimers and H-aggregates without the need of an external template.1 Such aggregates are fluorescence quenched due to fast relaxation from the upper to lower exciton states. Stable aggregates are formed by the dimeric carbocyanine dyes at micromolar concentration levels in sharp contrast to the behaviour of monomeric cyanine dyes. We found the dimeric carbocyanine dye aggregates to be disrupted in presence of certain quadruplex topologies.1 Duplex or single stranded DNA as well as other quadruplex conformations were unable to elicit the same response. While there are few reports on disruption of dye aggregates by specific biomolecules, there is a dearth of translation of these efforts towards live cell staining and imaging of specific cellular components.

 

 

  

Figure 1: Live-cell imaging of untreated human HeLa cells (i.e., no fixation, permeabilization or mounting steps) upon incubation with dye 1 (2 μM) for 24 h prior to cellular imaging (in phosphate buffer only).

 

Recent reports on the use of quadruplex-specific antibodies have demonstrated their efficacy in visualization of quadruplexes in cells.2, 3 However, such strategies are constrained by the availability of antibodies, multistep protocols and application on fixed and permeabilized cells. The dimeric carbocyanine dye reported by us is found to be potent in live cell imaging at low concentrations of usage and the application involves simple incubation. The staining profile of HeLa cells with the dimeric carbocyanine dye is interesting as bright fluorescence is clearly observed at the nuclear periphery and certain regions of the nucleus. These regions are suggested as corresponding to mitochondrial guanine-rich DNA.

 

Figure 2: Fixed-cell imaging of human HeLa cells upon incubation with dye 1 (2 μM) for 10 min prior to cellular imaging (in phosphate buffer only).

 

Bright fluorescent spots are also observed in fixed HeLa cells upon incubation with the dimeric carbocyanine dye. The guanine-rich rDNA present in the nucleoli have been suggested as adopting temporal quadruplex conformations during cellular processes.4-7 The bright fluorescent spots in fixed HeLa cells likely correspond to nucleolar rDNA.

Our dimeric carbocyanine dye is attractive for its ability to image quadruplex structures in cells and would be useful for tracking such structures during various cellular conditions.

 

References 

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