Synthetic threading intercalators as a new analytical probe for nucleic acid and gene detection

Abstract

Threading intercalator is a DNA ligand, whose major substituent group connected to the intercalating part is required to go through the adjacent base pairs of double-stranded DNA when the ligand becomes bound. The situation is typically fulfilled with the N, N'-disubstituted imide derivatives of naphthalene-1, 4, 5, 8-tetracarboxylic acid (N, N-disubstituted naphthalene diimide derivatives). In a complex with double-stranded DNA, the two substituents in a threading intercalator are projecting out in the major and minor grooves of DNA. This configuration results in kinetic stabilization of the formed complex, because the threaded bulky substituents can act as an anchor to prevent the ligand molecule from a facile dissociation from the DNA strand. We have been developing new analytical reagents for DNA by taking advantage of these peculiar characters of threading intercalation. As a typical threading design, three intrinsically intercalating molecular units were linearly connected by aminoalkyl chains to give trisintercalators. The resultant ligand seemed to prefer binding to the “labile” portion or sequence in the double stranded DNA where the strand is more susceptible to breathing or base-pair disruption. In another molecular design, thymine bases were introduced in the two N, N'-arms of naphthalene diimide. This ligand bound preferentially to a non-alternating continuous adenine sequence of single-stranded RNA due to the formation of hydrogen bonding between the thymine in the ligand and the adenine in the single strand RNA. Due to a similar A-T base pair forming interaction, this ligand stabilized the bulged duplex structure of oligonucleotide d(GCGAAACGC), for which the hairpin structure is more stable under ordinary solution conditions. The naphthalene diimide ligand carrying ferrocene parts on its N, N'-substituent arms allowed a highly sensitive electrochemical detection of double-stranded DNA. This provides a promising method for selectively detecting and quantitating target genes of interest. The principle is based on the redox current obtained from the ligand concentrated on the electrode, where the electrode-supported probe strand and the complementary target strand in solution hybridize to form a double strand. On the other hand, such a double stranded DNA-ligand complex on the electrode can be deemed as a pseudo-polyferroocene strand, since the ferrocene moieties are arranged along a single molecular chain of double-stranded DNA. This configuration of ferrocene units on the electrode gives a unique electrochemical behavior in enhancing the electron transfer between the reduced glucose oxidase and the electrode. Cyclic bis-threading intercalators are expected to form a doubly intercalated complex with double-stranded DNA offering a catenated structure. A cyclic naphtalene diimide derivative carrying a ferrocene unit within the macrocycle should find an application in the electrochemical detection of a base-mismatched DNA strand.