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Quantum dot-based molecular beacon to monitor intracellular microRNAs.

Lee J, Moon SU, Lee YS, Ali BA, Al-Khedhairy AA, Ali D, Ahmed J, Al Salem AM, Kim S - Sensors (Basel) (2015)

Bottom Line: In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon.The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal.Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bio-Medical Convergence, College of Medicine, Kwandong Catholic University, Gangneung-si, Gangwon-do 270-701, Korea. jonghwanlee104@gmail.com.

ABSTRACT
Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

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A schematic diagram of the R9-QD-miR124a beacons to image miR124a. The amine-terminated oligonucleotide containing the miR124a recognition sequence was annealed at the region with partial complementary sequences attached to the quencher molecules, which was designated as the miR124a-targeting oligomer. The carboxyl-terminated QD655 was conjugated with the miR124a-targeting oligomer and the R9 peptide. When the mature miR124a was hybridized with the miR124a binding region in the R9-QD-miR124a beacons, the quencher molecules were separated, resulting in an increase in the fluorescence signal.
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sensors-15-12872-f001: A schematic diagram of the R9-QD-miR124a beacons to image miR124a. The amine-terminated oligonucleotide containing the miR124a recognition sequence was annealed at the region with partial complementary sequences attached to the quencher molecules, which was designated as the miR124a-targeting oligomer. The carboxyl-terminated QD655 was conjugated with the miR124a-targeting oligomer and the R9 peptide. When the mature miR124a was hybridized with the miR124a binding region in the R9-QD-miR124a beacons, the quencher molecules were separated, resulting in an increase in the fluorescence signal.

Mentions: Here, we developed a new method for the fluorescence imaging of endogenous miRNAs using a signal-tunable molecular beacon technique based on QDs in living cells. To improve cellular uptake, a 9-mer arginine-rich peptide was attached to QDs. We targeted miRNA124a, which is known to be specifically and highly expressed in neurons and to have a well-established expression profile during neuronal development [17]. In the absence of miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon that quenches the system and leaves QDs located in adjacent regions to each other (Figure 1). No fluorescence signal is observed in this situation. In contrast, the presence of miRNA124a separates the quencher molecules from the R9-QD-miR124a beacon, resulting in signal-on of a red fluorescence signal. The recovery of fluorescence signal is closely dependent on the target miR124a concentration because the extent of separation of the quencher molecule on the R9-QD-miR124a beacon is affected by the number of target miR124a.


Quantum dot-based molecular beacon to monitor intracellular microRNAs.

Lee J, Moon SU, Lee YS, Ali BA, Al-Khedhairy AA, Ali D, Ahmed J, Al Salem AM, Kim S - Sensors (Basel) (2015)

A schematic diagram of the R9-QD-miR124a beacons to image miR124a. The amine-terminated oligonucleotide containing the miR124a recognition sequence was annealed at the region with partial complementary sequences attached to the quencher molecules, which was designated as the miR124a-targeting oligomer. The carboxyl-terminated QD655 was conjugated with the miR124a-targeting oligomer and the R9 peptide. When the mature miR124a was hybridized with the miR124a binding region in the R9-QD-miR124a beacons, the quencher molecules were separated, resulting in an increase in the fluorescence signal.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4507685&req=5

sensors-15-12872-f001: A schematic diagram of the R9-QD-miR124a beacons to image miR124a. The amine-terminated oligonucleotide containing the miR124a recognition sequence was annealed at the region with partial complementary sequences attached to the quencher molecules, which was designated as the miR124a-targeting oligomer. The carboxyl-terminated QD655 was conjugated with the miR124a-targeting oligomer and the R9 peptide. When the mature miR124a was hybridized with the miR124a binding region in the R9-QD-miR124a beacons, the quencher molecules were separated, resulting in an increase in the fluorescence signal.
Mentions: Here, we developed a new method for the fluorescence imaging of endogenous miRNAs using a signal-tunable molecular beacon technique based on QDs in living cells. To improve cellular uptake, a 9-mer arginine-rich peptide was attached to QDs. We targeted miRNA124a, which is known to be specifically and highly expressed in neurons and to have a well-established expression profile during neuronal development [17]. In the absence of miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon that quenches the system and leaves QDs located in adjacent regions to each other (Figure 1). No fluorescence signal is observed in this situation. In contrast, the presence of miRNA124a separates the quencher molecules from the R9-QD-miR124a beacon, resulting in signal-on of a red fluorescence signal. The recovery of fluorescence signal is closely dependent on the target miR124a concentration because the extent of separation of the quencher molecule on the R9-QD-miR124a beacon is affected by the number of target miR124a.

Bottom Line: In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon.The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal.Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bio-Medical Convergence, College of Medicine, Kwandong Catholic University, Gangneung-si, Gangwon-do 270-701, Korea. jonghwanlee104@gmail.com.

ABSTRACT
Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

Show MeSH
Related in: MedlinePlus