Rapid, multiplexed detection of the let‐7miRNA family using γPNA amphiphiles in micelle‐tagging electrophoresis

Abstract

miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base. In this report, we demonstrate an independent detection seven members of the let‐7 family of miRNA in a single run. Key to success is the use of mini‐PEG‐substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre‐programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag‐tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10–100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length.

Perspectives on conformationally constrained peptide nucleic acid (PNA): insights into the structural design, properties and applications

“…γPNA amphiphiles have been used in combination with fluorescent DNA nanotags in micelle-tagging electrophoresis. 284,285 The two short probes bound to adjacent positions on the same DNA target cooperatively stabilize each other by base stacking without compromising the specificity as generally observed when only one longer probe sequence was used. This strategy allows multiplex detection of various members of the let-7 miRNA family at a single mismatch resolution with detection limits of 10–100 pM.…”

“…This strategy allows multiplex detection of various members of the let-7 miRNA family at a single mismatch resolution with detection limits of 10–100 pM. 285 Likewise, the use of γPNA allows an efficient hybridization chain reaction (HCR) via γPNA·γPNA duplex formation to be performed using very short hairpin probes with only 5 nt stem, 5 nt loop, and 5 nt toehold making the total length of the hairpin constructs only 20 nt, far shorter than the DNA-based constructs (typically 30–40 nt). The use of miniPEG γPNA solved the problem of the limited solubility of the PNA-derived cHCR products.…”

Discrimination of Multiple Homologous Sequences Based on DNA Logic Gate and Elemental Labeling Technology

“…However, the detection of miRNA may be interfered with by the miRNA homologous sequence in real samples. For example, the homologous sequences of let-7 family have high sequence similarity, and there are only one, two, or three base differences among them, which poses great difficulties for accurate identification of let-7 family sequences. − What is more, the information on multiple sequences related to diseases can improve the accuracy of disease diagnosis, − the reliable detection of miRNA homologous sequences is of great significance for gaining better understanding of the functions of miRNAs in a wide range of biological processes. The strategy for detection of single nucleotide variation (SNV) which can recognize a single base difference of sequences shows great potential in the detection of sequences with high homology.…”

Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers

“…We have also attached alkanes to DNA oligonucleotides by probe hybridization, giving a rapid separation of PCR products and rapid quantitation of several miRNAs in a closely related let-7 panel. 13,14 In these applications, it was essential to use probes made of modified peptide nucleic acids (PNA) 15 to give tight binding to targets and enable attachment of other oligonucleotides, when necessary. Much of the success of the MTE method relies on proper choice of micellar running buffer.…”

“…This “micelle-tagging electrophoresis (MTE)” method relies on the statistical fluctuation of micelle size to confer a uniform drag upon all fragments as required for high-resolution separations. , This was accomplished by covalently grafting 18-carbon n -alkanes to PCR primers prior to their enzymatic extension to provide a binding site for micelles. We have also attached alkanes to DNA oligonucleotides by probe hybridization, giving a rapid separation of PCR products and rapid quantitation of several miRNAs in a closely related let-7 panel. , In these applications, it was essential to use probes made of modified peptide nucleic acids (PNA) to give tight binding to targets and enable attachment of other oligonucleotides, when necessary. Much of the success of the MTE method relies on proper choice of micellar running buffer.…”