Programmed Transfer of Sequence Information into a Molecularly Imprinted Polymer for Hexakis(2,2′-bithien-5-yl) DNA Analogue Formation toward Single-Nucleotide-Polymorphism Detection

Abstract

A new strategy of simple, inexpensive, rapid, and label-free single-nucleotide-polymorphism (SNP) detection using robust chemosensors with piezomicrogravimetric, surface plasmon resonance, or capacitive impedimetry (CI) signal transduction is reported. Using these chemosensors, selective detection of a genetically relevant oligonucleotide under FIA conditions within 2 min is accomplished. An invulnerable-to-nonspecific interaction molecularly imprinted polymer (MIP) with electrochemically synthesized probes of hexameric 2,2'-bithien-5-yl DNA analogues discriminating single purine-nucleobase mismatch at room temperature was used. With density functional theory modeling, the synthetic procedures developed, and isothermal titration calorimetry quantification, adenine (A)- or thymine (T)-substituted 2,2'-bithien-5-yl functional monomers capable of Watson-Crick nucleobase pairing with the TATAAA oligodeoxyribonucleotide template or its peptide nucleic acid (PNA) analogue were designed. Characterized by spectroscopic techniques, molecular cavities exposed the ordered nucleobases on the 2,2'-bithien-5-yl polymeric backbone of the TTTATA hexamer probe designed to hybridize the complementary TATAAA template. In that way, an artificial TATAAA-promoter sequence was formed in the MIP. The purine nucleobases of this sequence are known to be recognized by RNA polymerase to initiate the transcription in eukaryotes. The hexamer strongly hybridized TATAAA with the complex stability constant K = k/k ≈ 10 M, as high as that characteristic for longer-chain DNA-PNA hybrids. The CI chemosensor revealed a 5 nM limit of detection, quite appreciable as for the hexadeoxyribonucleotide. Molecular imprinting increased the chemosensor sensitivity to the TATAAA analyte by over 4 times compared to that of the nonimprinted polymer. The herein-devised detection platform enabled the generation of a library of hexamer probes for typing the majority of SNP probes as well as studying a molecular mechanism of the complex transcription machinery, physics of single polymer molecules, and stable genetic nanomaterials.

Oligonucleotide Determination via Peptide Nucleic Acid Macromolecular Imprinting in an Electropolymerized CG-Rich Artificial Oligomer Analogue

“…Our ITC measurements confirmed higher stability of the G-C pairs of the functional monomers with PNA than that of the A-T pairs. 17 Nevertheless, we still successfully and independently from matrix effects discriminated GC-rich analyte from two-and three-nucleobase mismatches with the appreciably low LOD.…”

“…[8][9][10][11] To minimize drawbacks of the challenging control of the density and orientation of natural probes and time-consuming optimization of the solution conditions for analyte-probe hybridization, 12 several nucleic acid analogs were designed and synthesized, [13][14][15][16] and then used as the probes. [17][18][19] Among them, peptide nucleic acids (PNAs) were selected, because of their sequenceselectivity and high affinity to complementary DNA and RNA single strands. However, they often need backbone and nucleobase modifications in order to pre-organize 20 their conformation, and then increase stability and sequence selectivity in duplex formation.…”

“…26 By engaging the molecular imprinting in polymer strategy, we have recently developed a fast, cost-effective, and simple procedure of one-step synthesis of a new electropolymerized DNA analog probe. 17 Moreover, the hybridizing probe was simultaneously immobilized on the transducer surface in this procedure. Using this macromolecular imprinting, we improved the orientation of the probes, thus tuning their density, which in turn influenced the hybridization yield.…”

Overview of Piezoelectric Biosensors, Immunosensors and DNA Sensors and Their Applications

“…Molecularly Imprinted Polymers can serve also as a recognition tool for specific sequences of genetic information. Bartold and coworkers decided to prepare a tool for the recognizing of single nucleotide polymorphism by the imprinted polymers [ 48 ].…”

Advances in Molecularly Imprinting Technology for Bioanalytical Applications

“…QCM has a relatively rich history compared with other materials fabricated with MIPs, as the usage of QCM-biosensors can be certainly dated back to the 1980s. To date, the applications of QCM-MIP-biosensors have been widely used for different types of biological substances from micro-biological molecules like glucose [112], to macro-biological molecules, for instance, DNA [113], proteins [114,115,116], bacteria [117,118] and virus [119], furthermore it has been reported that whole cells can be also imprinted for QCM-MIP-biosensors [120,121].…”