Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection

Abstract

Triplex molecular beacons (tMBs) possess great potential in biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine–guanine–cytosine (C-G•C+) and thymine–adenine–thymine (T-A•T) as the triplex stem, in which both the Watson–Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.

DNA Assembly‐Based Stimuli‐Responsive Systems

“…Yang's group has achieved the detection of miRNAs by manipulating the strength of the stem‐binding in the triplex molecular beacon (tMB) using pH values (Figure 9b). [ 74 ]…”

“…Moreover, the C-G•C + tends to form under acidic pH and T-A•T tends to form under neutral pH (Figure 1b). [69][70][71][72][73][74] Therefore, their pH responsive range can be regulated by the reasonable configuration of two Hoogsteen base pairing, so as to realize the control and reconfiguration of the DNA structure.…”

“…[150] In addition to DNAzymes, azobenzene and triplex structures are also used in nucleic acid detection. [74,256] Yuan's group designed a 3D DNA nanostructure based on the azobenzene-functionalized DNA nippers, which could realize the photoresponsive capture and release of the microRNAs (miRNAs). [256] The electrochemiluminescence (ECL) detection of miRNAs could be realized by the modification of ECL emitters Ru(bpy) 2 2+ and the quencher Alexa Fluor (AF).…”

Biomarker sensing platforms based on fluorescent metal nanoclusters

“…Although the use of biomarkers in cancer is very extensive due to the need for diagnosis in the early stages of the disease, there are many other health conditions for which patients can take advantage of an earlier diagnosis (Table 2). Furthermore, the strategies to follow are the same, for example the use of DNA [83][84][85] or aptamers 86 for the scaffolding of AgNCs that label a target miRNA when it is hybridized or the use of substrate molecules as stabilizing agents for the detection of enzyme activity. Interestingly, the use of uorescent NCs combined with an elegant mechanism of molecular recognition allows detecting not only internal factors such as genetic expression or enzymatic activity, but also a parasitic infection, such as malaria.…”

Coupling smartphone and CRISPR–Cas12a for digital and multiplexed nucleic acid detection

“…Recently, various methods have been developed for the nucleic acid detection, including fluorescent, 14 colorimetric, 15 and electrochemical 16 biosensors. Polymerase chain reaction (PCR) is still considered the standard of care for nucleic acid detection due to its high sensitivity and specificity.…”