A Pyrazine-based Fluorescence-enhancing Ligand with a High Selectivity for Thymine in AP Site-containing DNA Duplexes

Abstract

Studies on the chemistry of DNA-binding drugs and/or lowmolecular-weight ligands are of on-going interest due to their promising functions and biological activities, including their anti-cancer properties and ability to regulate gene expression. [1][2][3][4] Of particular interest to us is the development of a class of ligands suitable for gene detection, especially for singlenucleotide polymorphisms (SNPs) typing. 5 We have recently found a series of aromatic ligands that can selectively bind to a nucleobase opposite to an abasic site (AP site) in DNA duplexes, [6][7][8][9][10][11][12][13][14][15][16] and have proposed a new strategy of ligand-based fluorescence assay for SNPs typing. In contrast to typical DNA-binding ligands capable of targeting doublestranded DNAs by intercalation or groove binding, 17,18 it is a characteristic of our ligands to target non-Watson-Crick basepairing sites in DNA duplexes, where the binding is promoted by a pseudo-base pairing along the Watson-Crick edge of intrahelical target nucleobases. Fluorescence signaling is thus obtained by direct interactions between ligands and target nucleobases, and the SNP genotype of samples can be clearly distinguished by combining ligands with selectivity for respective target nucleobases. However, our assay is based on the monitoring of binding-induced fluorescence quenching of ligands: as has been observed for fluorescent nucleotide analogs, such as 2-aminopurine, 19 the fluorescence of ligands is significantly quenched due to stacking with nucleobases flanking the AP site. Further efforts are therefore necessary to develop ligands that exhibit fluorescence-enhancement and/or ratiometric emission responses, in order to make an assay more suitable for practical use.In this work, we successfully discovered a class of light-up ligands upon binding to AP site-containing DNA duplexes. The present ligand, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC, Fig. 1), is capable of selectively binding to thymine over other nucleobases, and the binding event is accompanied by a significant enhancement of its fluorescence. While the sensing function depends on nucleobases flanking the AP site, the response of DCPC is almost specific to the thymine base, making it possible to detect thymine-related mutations present in polymerase chain reaction (PCR) amplification products. Figure 2 shows typical fluorescence responses of DCPC (10 mM) to 23-mer AP site-containing DNA duplexes (5¢-GTGTG CGTTG ANA TGGAC GCAGA-3¢/3¢-CACAC GCAAC TXT ACCTG CGTCT-5¢, X = AP site; Spacer-C3, N = target nucleotide), as measured in solutions buffered to pH 7.0 (I = 0.11 M, at 5˚C). As can be seen from Fig. 2, the response strongly depends on nucleobases opposite the AP site. In the absence of DNAs, the ligand shows a very weak emission with a maximum at 412 nm, and almost no response is observed for guanine, even in the presence of 5 equiv. of DNA (50 mM). Similarly, the response of DCPC is only moderate for adenine and cytosine. By contrast, DCPC exhibits a significant enhancemen...

Recent Progress in Abasic Site-binding Small Molecules for Detecting Single-base Mutations in DNA

“…The observed sensitivity is almost comparable to that of unamplified imaging SPR based on DNA or RNA hybridization adsorption (detection limit: 10 nM for 18-meric oligos), 32 and it is superior to that of unamplified scanning SPR for DNA detection (detection limit: 150 nM for 143-meric/101-meric duplex). 31 It is also of interest to note that this sensitivity has not been obtained in homogeneous solutions for the 3,5-diaminopyrazines-DNA binding, 19,20 where an effective fluorescent response was obtained in the concentration range roughly equivalent to the dissociation constant (~μM). The relatively high sensitivity we obtained in the present heterogeneous assay may be assisted by an inherent property of the SPR system to sense a change in surface mass concentration on the sensor chip (1000 RU ≈ 1 ng/mm 2 for most biomolecules), 30 and a binding of higher molecular weight analytes would give a larger response in RU.…”

“…in DNA duplexes, [10][11][12][13][14][15][16][17][18][19][20][21] and have proposed a new strategy of ligand-based fluorescence assay for SNP typing. In contrast to typical DNA-binding ligands capable of targeting double stranded DNAs by intercalation or groove binding, 22,23 it is characteristic of ligands to bind to non-Watson-Crick basepairing sites in DNA duplexes, where the binding is promoted by a pseudo-base pairing along the Watson-Crick edge of intrahelical target nucleobases, and also by stacking with two nucleobases flanking the AP site.…”

“…Useful affinity and selectivity for target nucleobases opposite the AP site has been indeed obtained by various kinds of heterocyclic planer compounds, including cytosine-selective 2-amino-1,8-naphthyridine derivatives, [10][11][12][13][14] guanine-selective pterin derivatives, [15][16][17] adenineselective 6,7-dimethyllumazine, 18 and thymine-selective 3,5-diaminopyrazine derivatives. [19][20][21] All of these ligands show a complexation-induced fluorescence signaling, and genotype of samples can be clearly distinguished by combining ligands with selectivity for respective target nucleobases.…”

Enhancement in fluorescence response by a quencher for amiloride upon binding to thymine opposite an abasic site in a DNA duplex

“…Fluorophores are usually covalently labeled at the end of an ODN strand [2,3] or within an ODN strand intrahelically [4,5]. In contrast to these fluorophore-labeled ODN probes, we have proposed a new label-free SNPs typing method based on apurinic or apyrimidinic (AP or abasic) site-containing ODN probes in combination with fluorescent ligands that can discriminate a specific nucleobase over the other three nucleobases [6][7][8][9][10][11][12][13][14]. In our method, synthetic and/or biotic fluorescent ligands bind to the intrahelical target nucleobases at the AP site by forming a pseudo-base pairing between ligands and the target nucleobases, accompanied by fluorescence signaling.…”

“…In our method, synthetic and/or biotic fluorescent ligands bind to the intrahelical target nucleobases at the AP site by forming a pseudo-base pairing between ligands and the target nucleobases, accompanied by fluorescence signaling. We have successfully discovered a series of fluorescence ligands that can recognize a target nucleobase with high affinity and selectivity at the AP sites in DNA duplexes, including cytosine (C)-selective naphthyridine derivatives [6][7][8], guanine (G)-selective pteridine derivatives [9], adenine (A)-selective alloxazine [10] and lumazine derivatives [11], and thymine (T)-selective amiloride [12,13] and flavin derivatives [14].…”