22AG G-quadruplex RNA/QnMorpholine-mediated fluorimetric detection of miR-21.

Herein we report a simple ligation/transcription-mediated system, using a 22AG G-quadruplex RNA secondary structure and a fluorescence-inducing QnMorpholine probe, for the detection of miR-21. In the presence of the target miR-21, two oligonucleotide probes (promoter and reporter) were ligated, thereby transcribing the 22AG RNA sequence, a complement of the reporter probe. In contrast, in the absence of this target-induced ligation, the reporter complement could not be transcribed to produce the 22AG RNA sequence. Subsequent addition of the QnMorpholine probe resulted in binding with the 22AG G-quadruplex RNA, thereby generating high fluorescence; no fluorescence occurred in the absence of this secondary structure. Hence, the presence of miR-21 was evidenced by a target-induced high-intensity signal. This simple one-pot fluorimetric system, which could detect miR-21 of up to 3.08 femtomolar in less than 30 min, holds promise as a diagnostic tool for selective and sensitive miRNA detection.

Directly arylated oligonucleotides as fluorescent molecular rotors for probing DNA single-nucleotide polymorphisms.

We developed direct arylated oligonucleotide based molecular rotor (AOMR) to discriminate perfect matched DNA sequence from one base mismatched sequences. Quinolinium salts attached to vinyl aniline would be excellent fluorescent analogs with molecular rotor properties and are suitable for the detection of microenvironment change arising from dynamic motions with match-mismatch DNA base pairs. We applied direct N6 arylation of the adenosine located in natural oligonucleotide as a tool to incorporate the molecular rotor (Quinolinium salts attached vinyl aniline) and used it to discriminate perfect matched DNA sequence from one base mismatch sequences. The fluorescence and quantum yield of arylated oligonucleotide based molecular rotor (AOMR), particulary, RMAQn reveals 28.3 times higher discrimination factor with perfect matched sequence (RMAQn:T) (QY = 0.17) compare to single strand RMAQn (QY = 0.006) and one base mismatched sequence (RMAQn:G, RMAQn:A, and RMAQn:C) at λmax = 600 nm (orange emission), which would be useful for in vivo application. RMAQn:T duplex also showed high brightness (6068), 32.9 times higher than single strand RMAQn (192), as a result of restricted rotation of the Quinolinium salts attached vinyl aniline on adenosine moiety with perfect matched sequence compare to the mismatch sequences. Arylated oligonucleotide based molecular rotor (AOMR) proves to be an unprecedented sensitivity in detecting local dynamics of nucleic acids and also would be simple and cost-effective method to prepare SNP probe.

Highly sensitive, selective, and rapid detection of miRNA-21 using an RCA/G-quadruplex/QnDESA probing system.

In this study we developed a very simple and rapid miRNA 21 detection system using a novel quinolinium diethylamino salicylaldehyde (QnDESA) probe for sensing the 22AG hybrid G-quadruplex with a single-step rolling circle amplification (RCA) reaction. We synthesized a circular DNA padlock template containing a sequence complementary to the 22AG hybrid G-quadruplex, used SplintR ligase to ensure perfect hybridization with miRNA 21, applied this circular DNA and phi-29 DNA polymerase for tandem amplification of the 22AG hybrid G-quadruplex sequence, and then probed the product using QnDESA. This combination of RCA-G-quadruplex and QnDESA allowed the rapid (1 h) and simple one-pot detection of miRNA 21 based on a change in fluorescence. In addition, this system displayed high sensitivity (limit of detection: 1.37 fM) and selectivity. This probing system should also be useful for identifying a diverse range of DNA- and RNA-based biomarkers.

RNA Polymerase-Mediated Stepwise RNA-Primed RNA Polymerization for Site-Specific Multiple Labeling into RNA: A Fluorescence Resonance Energy Transfer Probe Detects the Structural Change of an RNA G-Quadruplex.

In this paper, we report a stepwise RNA-primed RNA polymerization method for the site-specific incorporation of multiple fluorescent moieties into RNA, mediated by an RNA polymerase. A screen of several RNA polymerases revealed that T7 RNA polymerase was the only one that functioned in the RNA-primed RNA polymerization. In the first fluorescence labeling step, a fluorescent rUthioTP residue was incorporated directly into the RNA using T7 RNA polymerase; the second fluorescence labeling step was performed using a post-labeling strategy: directly introducing an rUamiTP residue into RNA, using T7 RNA polymerase, and then reacting with ylidenemalononitrile enamine (P3). The whole process for the site-specific introduction of the multiple labeled moieties was performed through stepwise RNA-primed RNA polymerization. Interestingly, the resulting multiple-labeled RNA exhibited fluorescence resonance energy transfer (FRET) between the two fluorescent labels in the RNA. We optimized the FRET-breaking point in the RNA by changing of distance between the two fluorescent labels and then used this property for the detection of the structural change of the RNA. The FRET signal increased in intensity upon the transformation of the RNA from a single-strand structure to the G-quadruplex. This approach for site-specific FRET labeling into RNA using RNA polymerase suggests the possibility of performing other diverse site-specific modifications at predefined positions in RNA.

Polymerase-mediated synthesis of p-vinylaniline-coupled fluorescent DNA for the sensing of nucleolin protein-c-myc G-quadruplex interactions.

In this paper we report the synthesis of two deoxyuridine derivatives (dUCN2, dUPy)-featuring p-vinylaniline-based fluorophores linked through a propargyl unit at the 5' position-that function as molecular rotors. This probing system proved to be useful for the sensing of gene regulation arising from interactions between this G-quadruplex and nucleolin.

Direct and selective metal-free N6-arylation of adenosine residues for simple fluorescence labeling of DNA and RNA.

We have developed an unprecedented transition metal-free approach for the direct fluorescence turn-on labeling of natural oligonucleotides through selective N6-arylation of adenosine moieties. This method allows the simple and direct fluorescence labeling of natural unmodified DNA and RNA, but is dependent on the secondary structure, favoring single-stranded structures.

rkDNA-graphene oxide as a simple probe for the rapid detection of miRNA21.

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate-containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA-GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.

Highly fluorescent morpholine naphthalimide deoxyuridine nucleotide for the detection of miRNA 24-3P through rolling circle amplification.

In this study we synthesized the nucleotide dUrkTP, a highly fluorescent naphthalimide deoxyuridine triphosphate that undergoes aggregation-induced emission (AIE). We incorporated and extended dUrkTP during the primer extension of DNA mediated by DNA polymerase, and also in the rolling circle amplification of DNA mediated by Phi29 polymerase. Accordingly, we could use this fluorescent nucleotide for the detection of microRNA 24-3P, a biomarker of porcine reproductive and respiratory syndrome virus. The direct labeling system obtained during rolling circle DNA amplification exhibited increased fluorescence, due to AIE of the dUrkTP residue upon gel formation, thereby allowing the detection of miRNA 24-3P. This direct labeling system facilitated the simple and inexpensive detection of miRNA 24-3P with high sensitivity (limit of detection: 3.58 fM) and selectivity.