Spectroscopic Analyses of the Noncovalent Self-Assembly of Cyanines upon Various Nucleic Acid Scaffolds

Abstract

Noncovalent forces are involved in the supramolecular selfassembly. [1][2][3][4] We previously showed that a class of chromophores known as cyanines were capable of spontaneously selfassembling upon a variety of biopolymers including carbohydrates. [5][6][7][8] Cyanines are photosensitive dyes composed of two quaternized, nitrogen-containing, and heterocyclic ring structures, that are linked by a polymethine bridge. 9 Cyanines are characterized by high molar absorptivity, low intrinsic fluorescence and large fluorescence enhancement following self-assembly upon various templates. Supramolecular selfassembly involves reversible, noncovalent, and electrostatic (coulombic) interactions including hydrogen and/or charge bonding, van der Waals and hydrophobic forces. Cyanine selfassembly is sometimes accompanied by strong, sharp and bathochromic fluorescence, characteristic of J-aggregates. 10Cyanines are also capable of forming hypsochromic, nonfluorescent or very weakly fluorescent H-aggregates. 10 Controlling and tuning the supramolecular self-assembly processes will offer insights to enable programmability to achieve desired functional nanomaterials. We therefore further investigated the self-assembling of cyanines from our molecular "library" in order to identify a chromophore that was capable of forming intensely fluorescent J-aggregates.Our objectives were: 1) Identify cyanine chromophores from our molecular library that would be a sensitive probe for nucleic acids; 2) Correlate the form-function aspects of cyanine chemistry to self-assembly on nucleic acid scaffolds; 3) Compare the detection limits of single-stranded viral DNA, double-stranded viral DNA, double-stranded genomic DNA, and RNA, using self-assembly; 4) Optimize detection conditions for nucleic acids based upon supramolecular self-assembly including the stability of the nucleic acids-cyanine complex; and 5) Demonstrate supramolecular self-assembly processes were involved in the aggregation of the cyanine upon nucleic acid scaffolds. Results from these investigations are described in this paper. Experimental Reagents and chemicalsThe chemical structures of various cyanines from our molecular "library" 5,11 are shown in Fig. 1. Stock solutions of cyanines were prepared in methanol and stored refrigerated. Single stranded, circular, fX174 virion DNA (5386 bases, Mr = 1.7 ¥ 10 6 Da) from fX174 am3 cs70 bacteriophage was purchased from New England Biolabs (Ipswich, MA). Greater than 85% of the DNA molecules were circular. The double stranded bacteriophage lDNA composed of 48502 bp (Mr = 3.2 ¥ 10 7 Da) was from Promega (Madison, WI). Unsheared Escherichia coli (E. coli) genomic DNA with an average size of 16 kb (Mr = 1.4 ¥ 10 7 Da) was purchased from Sigma Aldrich (St. Louis, MO). The DNA had been purified by equilibrium buoyant density gradient ultracentrifugation in CsCl and agarose gel (0.8%) electrophoresis was used for size determination. Genomic DNA was dissolved using TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM NaCl and 1 mM EDTA). The DNA solution wa...

Supramolecular Self-Assembling Cyanine as an Alternative to Ethidium Bromide Displacement in DNA-Drug Model Interactions during High Throughput Screening

“…7 The concentration of polyamine inhibiting cyanine binding by one-half (50% drop in fluorescence from the DNA-cyanine J-aggregate) was designated as IC50. The association constant (Ka) was calculated from the reciprocal of the IC50.…”

“…7 The polyamine spermine [C10H26N4] (98% pure; moisture, 0.33%; Mr = 202.3) was from MP Biomedicals (Solon, OH). The FTIR spectrum of spermine conformed to standard.…”

“…5 One method to screen for DNA-drug interactions is "dye displacement," whereby dyes exhibit bright fluorescence upon binding to DNA that is followed by fluorescence loss upon dye displacement by the drug. 2,6 Dyes such as PicoGreen, SYBR Green I, cyanine 7 (and references therein), ethidium bromide (EB), Hoescht 33258 and 4,6-diamidino-2-phenylindole, 2 all bind to DNA accompanied by varying levels of fluorescence enhancement relative to the unbound fluorophore. 2 For example, EB fluorescence increases 24-fold upon binding to DNA and Hoescht 33258 undergoes 140-fold enhancement.…”

“…8 As an alternative to EB, we report a cyanine dye that spontaneously self-assembled upon DNA scaffolds accompanied by intense fluorescence from the J-aggregate. 7 We used DNA-spermine (polyamine) binding 9 as a model for DNA-drug interactions. [2][3][4] We describe various factors influencing the competition between cyanine and polyamine for binding to DNA and a 10-fold potentiating effect of methanol on binding.…”

“…Positive (100%) control was DNA + cyanine 7,11 and DNA + polyamine + cyanine represented competitive inhibition. Negative controls had fluorescence <10% of maximal fluorescence from the DNA-cyanine ensemble.…”