Light‐Up Hoechst–DNA Aptamer Pair: Generation of an Aptamer‐Selective Fluorophore from a Conventional DNA‐Staining Dye

Abstract

We have designed a strategy to generate a light-up fluorophore-aptamer pair based on a down-modification of a conventional DNA-staining dye to suppress its affinity to the original dsDNA targets, followed by reselection of aptamers that would bind to the modified dye. Following this line, we prepared a micropolarity-sensitive Hoechst derivative possessing two tBu groups with low affinity to the usual AT-rich dsDNA targets. DNA aptamers selected in vitro from a random pool worked as triggers to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative, and the shortened 25-mer sequence showed remarkable enhancement (light-up). The 25-mer sequence was split into binary aptamer probes, thus enabling us to detect a target nucleic acid sequence with a single-nucleotide resolution by use of unmodified DNA as a probe.

Design of Functional Nucleic Acid Systems for Biomolecular Analysis

“…45,46 Following the evolution process shown in Figure 5, we successfully obtained a new fluorescent DNA composed of a modified Hoechst dye and an optimized 25-mer DNA-tag structure (Figure 6a). 40 The otherwise almost nonfluorescentmodified Hoechst dye, having tert-butyl groups on its benzene ring to suppress binding to the original dsDNA target, enhanced its fluorescence by 191 times on binding with the optimized DNA sequence (K d = 878 nM). It is interesting, and even surprising, that the simple in vitro selected DNA sequence can work as a trigger to light up the otherwise nonfluorescent fluorophores on binding.…”

New Ideas for in Vivo Detection of RNA

“…www.intechopen.com 3.3 Novel approaches for "light-up" dye/aptamer pairs selection Sando and coworkers suggested a new route for generating specific light-up pairs starting with a conventional fluorescent DNA-staining dye that exhibits a number of favorable characteristics, including cell permeability, solubility, and stability in cellular environments (Sando et al, 2007;Sando et al, 2008). The key idea to this approach was demonstrated using Hoechst dye 33258, a widely used fluorescent stain for dsDNA molecules that fluoresces upon binding to the minor groove of dsDNA.…”

“…The most effective modification of the dye for this purpose was the addition of two t-Butyl groups to its terminal phenol ring (The resulting " Hoechst 33258 derivative" is shown in Figure 2C). Next, SELEX was used to obtain a DNA aptamer that bound Hoechst 33258 derivative with K d = 878 nM and enhanced fluorescence emission by 191-fold (Sando et al, 2007). Further work obtained an improved RNA aptamer showing higher affinity for the dye (K d =35 nM) but somewhat reduced emission enhancement (56-fold).…”

“…A 20-fold fluorescence enhancement was reported for this biosensor in the presence of 2 μM concentration of the target, with discrimination factors greater than 20-fold for half of the sequences differing from the intended analyte by single nucleotide substitutions. Sando and coworkers tested the applicability of Kolpashchikov's strategy to design a binary probe using their novel light-up Hoechst derivative/ DNA aptamer light-up pair (see section 2.2 above) (Sando et al, 2007). The same design principle was used: the DNA aptamer for the Hoechst dye was split into two molecules, each of which contains half of the sequence complementary to the target.…”

Structure and Function of Molecular Assembly. A Personal Reminiscence

“…It is composed of two essential processes, selection of random sequences on the target by using in the present case, targetimmobilized magnetic particles and PCR (polymerase chain reaction) amplification of the binding sequences (aptamers). By repeating the selection/amplification cycles, one can exponentially enrich or evolve the strongly binding aptamers with K in the order of 10 6 and 10 8 M ¹1 for the DNA 45 and RNA 46 aptamers, respectively. Partial sequences of the aptamers, in light of their computer-predicted secondary structures, were also checked in search for the true dye binding domain of the aptamers.…”