Secondary‐Structure‐Inducible Ligand Fluorescence Coupled with PCR

Abstract

Single-stranded DNA can change its structure dynamically in response to the presence of a complementary strand. The large structural change from the hairpin secondary structure to the double-stranded form leads to the chemistry of molecular beacons (MBs), or hairpin DNA oligomers having both a fluorescent and a quenching chromophore in the hairpin stem.[1] MBs can be used to report the presence of complementary strands by measuring the increasing fluorescence because of the decreasing energy transfer efficiency. Most DNA probes for fluorescent detection are covalently bound to fluorophores to make energy transfer effective. [1, 2] We have focused on an alternative way to link the fluorescence change with the structural changes of DNA that are induced during polymerase chain reaction (PCR) amplification.[3] We describe herein the chemical concept of noncovalent fluorescent DNA labeling by ligand binding to the secondary structure, which allows us to monitor PCR progress by measuring the change in fluorescence emitted from ligand-primer complexes in a homogeneous solution. This concept is not only complementary to that using fluorescent dye bound selectively to PCR product duplexes, [4] but it also expands the possibility of real-time PCR.The concept of DNA labeling by secondary-structureinducible ligand fluorescence is shown in Figure 1. PCR primers are labeled with a hairpin tag containing cytosine bulges (C-bulges). The molecule 2,7-diamino-1,8-naphthyridine (DANP) binds to a C-bulge in its protonated form (DANPH + ) and emits fluorescence at 430 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP.[5] We hypothesized that, as the PCR progresses, the hairpin tag will dissolve and be transformed into a duplex, thus resulting in the loss of the DANP binding site and a decrease in the fluorescence at 430 nm. Toward this end, we investigated the hairpin tags. These tags should identify the DANP binding site without disturbing the fluorescence efficiency, should not interfere with the PCR, should be transformed effectively into the duplex during PCR, and should be applicable to diverse primers. First, the sequence flanking the C-bulge producing the highest fluorescence intensity was investigated by measuring DANP fluorescence with duplexes having a C-bulge flanked by A-T and T-A base pairs. The G-C and C-G base pairs were omitted from the flanking base pairs because of their quenching of DANP fluorescence.[5b] The characteristic spectrum with a broad single peak at 430 nm was obtained for the A_A/TCT sequence. The relative fluorescence intensity at 450 nm for the four C-bulge duplexes and a fully matched duplex are shown in Figure S1 in the Supporting Information. The C-bulge in the A_A/TCT sequence enhanced DANP fluorescence by 7.6-fold compared with the fully matched duplex and by 82-fold compared with free, unbound DANP.We then designed hairpin tags comprising one to three A_A/TCT units separated by three to five base pairs and a T4 hairpin loop (Table 1). The intensity of DANP fluores...

Naphthyridine tetramer with a pre-organized structure for 1:1 binding to a CGG/CGG sequence

“…Dimeric naphthyridine derivatives bind selectively to a GG mismatch, where each of the two naphthyridine moieties recognizes two mispaired guanine bases (13,18). While the mismatch binding ligand (MBL) has been studied as a diagnostic tool for SNPs (13,14), trinucleotide repeats (15–17), telomeric repeats (20,21) and as a molecular glue for controlling DNA hybridization (31–35), MBLs with enhanced selectivity and affinity are still needed for practical applications.…”

RT‐Hpro‐PCR: A MicroRNA Detection System Using a Primer with a DNA Tag

“…The Hpro can either form a secondary hairpin structure containing a C‐bulge or hybridize with the tag sequence to create a fully matched Hpro‐tag duplex. When the DANP binds the C‐bulge, the DANP ⋅ C‐bulge complex emits fluorescence at approximately 430–450 nm, with a 30 nm bathochromic shift from the fluorescence of the free, unbound state . The open form of the probe with the primer tag and the hairpin structure of the probes with DANP are metastable at room temperature; in other words, this PCR system monitors the deflection of the equilibrium between the Hpro and the Hpro associated with the tag of the primer duplex.…”

Zn2+-cyclen-based complex enable a selective detection of single-stranded thymine-rich DNA in aqueous buffer

“…To improve the selectivity, new strategies for DNA detection are in urgent demand. As we know, base pairing is highly specific, and thus some probes that could selectively pair with nucleic acid bases with the combination of π-π stacking of neighboring base pairs were developed for recognizing some nucleic acid motifs such as bulges (Takei et al, 2009;Sato et al, 2012) and mismatches (Nakatani et al, 2001;Kobori et al, 2004;Sato et al, 2011). Besides, base pairing was also incorporated in some chromophores for DNA-templates assemblies (Janssen et al, 2007(Janssen et al, , 2009a(Janssen et al, , 2009b(Janssen et al, , 2010 or fluorescent detection of adenine-rich single-stranded DNA (ssDNA) (Lou et al, 2014).…”