z Medicinal Chemistry & Drug Discovery Selective Binding of Thioflavin T in Sequence-Exchanged Single Strand DNA Oligomers and Further Interaction with Phospholipid Membranes Shrabanti Das and Pradipta Purkayastha* [a] Selectivity of ThT towards sequence exchanged single strand DNA (ssDNA) oligomers in absence and presence of surface charged lipid vesicles has been studied in detail. The dye shows special affinity for TCTTA sequence with its –N(CH 3 ) 3 structural counterpart and p-interaction with adenine in 5’- CCCTCTTAACCC-3’ (TC4). However, fluorescence spectroscopy and molecular docking suggest that the dye is perfectly aligned with the TAA sequence in 5’-CCCTTAATCCCC-3’ (TT4). Inter- actions with cationic and anionic lipid vesicles were studied to understand the biological relevance in specificity of ThT bind- ing to ssDNA oligomers. Fluorescence intensity of ThT increases significantly due to interaction with charged liposomes along with a considerable hypsochromic shift. Interestingly, evolution of a shoulder at 465 nm was observed solely in cationic lipid membranes due to ThT-dimer formation. It is concluded that the ssDNA bound ThT penetrates the hydrophobic bilayer of the anionic vesicles. However, they attach superficially to the cationic vesicles facilitating dimer formation. Exchange of the nucleotide sequence in ssDNA led to change in the excited state lifetime of bound ThT in anionic liposomes. Introduction Nucleic acid is an oligomer formed of chain-linked nucleotides encoding genetic blueprint of organism. Important genetic functions, such as, gene transcription, [1] mutagenesis, [2] gene expression in cells [3, 4] etc. are carried out by DNA from one generation to the other. [5, 6] Single-stranded oligonucleotides or aptamers bind to targets with high affinity and selectivity and help in biosensing, diagnostics, and therapeutics. Moreover, aptamers can reversibly denature on changing the environ- mental parameters like temperature, pH, electric potential, etc. They simply unfold and can be revived as folded to regain the original binding conditions. [7] Same is true for simple ssDNA oligos. Small molecules play important roles in many biological processes because of easy diffusion across cell membrane. [8] In cells, small molecules help in cell signaling, act as pigments, and take part in defense mechanisms. [9] Usually, they bind to DNA strand by three types of interactions: (i) electrostatic, (ii) groove binding and (iii) intercalation within base pairs. Affinity, strength and mode of such binding are controlled by the structure and electrostatic nature of both host and guest. [10] Although no specific binding mode has been established till date, generally the oligomers bind planar molecule in inter- calative mode between the bases, whereas flexible and crescent shaped molecules prefer groove binding. [11,12] Aptamers are considered to be ideal molecular recognition probes for small molecules due to high degree of selectivity compared to antibodies. [13, 14] They mimic properties of anti- bodies in context to molecular recognition in spite of their biological difference. Target proteins for ssDNA are easier to discover compared to small molecules targeting nucleic acids because of lack in spatial structural information for the nucleic acids. Hence, research on finding small molecules targeting protein does not completely fulfil the requirements for targeting the nucleic acids. [15] Aptamers and/or ssDNA oligos are used in wide range of applications, such as medicine, cell biology, and analytical chemistry. These have potential uses in drug development, cell tracking and microbial detection and are comparable to antibodies. [16] Specific recognition of DNA sequences by small molecules is of importance both for targeting specific genes in a genome, and for chemotherapeu- tic purposes. [17, 18] Extensive research on minor groove targeting small molecules that are highly sequence-specific have con- tributed much toward DNA damage studies. For example, sequence-specific oligomers, such as, hairpin pyrrole–imidazole polyamides bind to the minor groove of DNA. [19] While groove binding or intercalation may lead to DNA unwinding, it becomes necessary to understand the binding mode of small molecules to ssDNA to a particular sequence of nucleotides. Hence, specific DNA sequences can be chosen in order to explore the selectivity in binding of small molecules. Information on selective binding helps to know the detailed mechanism of interaction of the drug within biological cell. With this intention, we chose three single strand oligos (aptamers) of 12 bases each: 5 0 -CCCCCCCCCCCC-3 0 ðCC4Þ [a] S. Das, Dr. P. Purkayastha Department of Chemical Sciences Indian Institute of Science Education and Research (IISER) Kolkata Mohanpur 741246, WB, India E-mail: ppurkayastha@iiserkol.ac.in Supporting information for this article is available on the WWW under https://doi.org/10.1002/slct.201700194 Full Papers DOI: 10.1002/slct.201700194 5000 ChemistrySelect 2017, 2, 5000 – 5007  2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim