A series of structurally similar 1-substitued heteroaryl fluorene derivatives were prepared in a simple single step reaction, oxidized to fluorenones and then both, fluorenes and fluorenones, were methylated to enhance the solubility and increase the affinity to DNA/RNA. Interactions of both, fluorene and fluorenone analogues with various ds-DNA, ds-RNA revealed strong ds-DNA/RNA binding, and various thermal stabilization effects. Most intriguingly, some fluorene derivatives showed opposite fluorescence change (increase for ds-RNA and decrease for ds-DNA), which was not previously reported for any fluorene analogue. CD experiments along with other methods support ds-DNA minor groove binding and major groove ds-RNA binding. All compounds showed negligible interaction with G-quadruplex DNA. Very low cell cytotoxicity of studied compounds combined with very efficient cellular uptake makes these fluorescent dyes safe for laboratory applications. Moreover, especially compounds which show opposite fluorescence response to ds-DNA and ds-RNA, are promising lead compounds for further studies aimed toward ds-RNA-specific fluorescence markers. (C) 2017 Elsevier Ltd. All rights reserved.