We newly synthesized thioflavin T (ThT) analogs for which the methyl group at the N3 position on the benzothiazole ring was replaced with either a ((p-(dimethylamino)benzoyl)oxy)ethyl group (ThT-DB) or a hydroxyethyl group (ThT-HE). In several neutral buffers, ThT-HE bound to a parallel guanine-quadruplex (G4) DNA and selectively emitted strong fluorescence at 74- to 240-fold higher intensities than those in the presence of double-stranded DNA (dsDNA), whereas ThT resulted in only 13- to 25-fold higher intensities. Furthermore, circular dichroism (CD) analyses using ThT, ThT-DB, and ThT-HE showed that these compounds could induce topological changes in G4. In addition, the different chemical structures of the N3 substituents could alter a G4-DNA conformation. These results indicate a great potential for N3-substituted ThT analogs as G4 probes and drug leads to achieve gene expression regulation.
Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of G-quadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation.
We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.
The field of care testing toward the analysis of blood
and saliva
lacks nowadays simple test techniques for biomarkers. In this study,
we have developed a novel nucleobase analog, Ugu, which
is a uracil derivative bearing a guanine base at the 5-position. Moreover,
we attempted the development of aptamers that can bind to secretory
immunoglobulin A (SIgA), which has been examined as a stress marker
in human saliva. It was observed that the acquired aptamer binds strongly
and selectively to the SIgA dimer (K
d =
13.6 nM) without binding to the IgG and IgA monomers of human serum.
Reduction of the aptamer length (41 mer) successfully improved 4-fold
the binding affinity (K
d = 3.7 nM), compared
to the original, longer aptamer (78 mer). Furthermore, the development
of a simple detection system for human saliva samples by fluorescence
polarization was investigated, using the reported human salivary α-amylase
(sAA) and the SIgA-binding aptamer. Comparison of the present method
with conventional enzyme-linked immunosorbent assay techniques highlighted
a significant Pearson’s correlation of 0.94 and 0.83 when targeting
sAA and SIgA, respectively. It is thus strongly suggested that a new
simple test of stress markers in human saliva can be quantified quickly
without bound/free (B/F) separation.
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