Promoting bioanalytical concepts in genetics: A TATA box molecularly imprinted polymer as a small isolated fragment of the DNA damage repairing system.

We demonstrate that a new, stable, artificial TATA (T - thymine, A - adenine) box is recognized by amino acids recognizing the natural TATA box. Here, the former mimicked, as a minimal motif, oligodeoxyribonucleotide interactions with amino acids of proteins involved in repairing of damaged dsDNA. By electropolymerization, we molecularly imprinted non-labeled 5'-TATAAA-3' via Watson-Crick nucleobase pairing, thus synthesizing, in a one-step procedure, the hexakis[bis(2,2'-bithien-5-yl)] TTTATA and simultaneously hybridizing it with the 5'-TATAAA-3' template. That is, a stable dsDNA analog having a controlled sequence of nucleobases was formed in the molecularly imprinted polymer (MIP). The 5'-TATAAA-3' was by the X-ray photoelectron spectroscopy (XPS) depth profiling found to be homogeneously distributed both in the bulk of the MIP film and on its surface. The 5'-TATAAA-3' concentration in the 2.8(±0.2)-nm relative surface area, ~140-nm thick MIP film was 2.1 mM. The MIP served as a matrix of an artificial TATA box with the TATAAA-promoter sequence. We comprehensively characterized this artificial DNA hybrid by the polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and X-ray photoelectron spectroscopy (XPS). Further, we examined interactions of DNA repairing TATA binding protein (TBP) amino acids with the artificial TATA box prepared. That is, molecules of l-phenylalanine aromatic amino acid were presumably engaged in stacking interactions with nucleobase steps of this artificial TATA box. The nitrogen-to­phosphorus atomic % ratio on the surface of the MIP-(5'-TATAAA-3') film increased by ~1.6 times after film immersing in the l-glutamic acid solution, as determined using the XPS depth profiling. Furthermore, l-lysine and l-serine preferentially interacted with the phosphate moiety of 5'-TATAAA-3'. We monitored amino acids interactions with the artificial TATA box using real-time piezoelectric microgravimetry at a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy under flow injection analysis (FIA) conditions.

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

We devised and fabricated a chemosensor for determination of the genetically relevant 5'-GCGGCGGC-3' (G = guanine; C = cytosine) oligonucleotide. For that, we simultaneously electrosynthesized and electrode-immobilized a sequence-defined octakis(2,2'-bithien-5-yl) DNA hybridizing probe using both a "macromolecular imprinting in polymer strategy" and a sequence-programmable peptide nucleic acid (PNA) template. With electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) transductions under stagnant-solution and flow injection analysis (FIA) conditions, respectively, we determined the above oligonucleotide with 200-pM EIS limit of detection. With its EIS-determined apparent imprinting factor of ∼4.0, the chemosensor was discriminative to both mismatched oligonucleotides and Dulbecco's modified Eagle's medium sample interferences.

Molecularly Imprinted Polymer Chemosensor for Selective Determination of an N-Nitroso-l-proline Food Toxin.

A molecularly imprinted polymer (MIP)-based chemosensor for the selective determination of a chosen toxin, N-nitroso-l-proline (Pro-NO), was devised and fabricated. By means of DFT, the structure of the pre-polymerization (functional monomer)-template complex was modeled. This complex was then potentiodynamically electropolymerized in the presence of cross-linking monomer to form a MIP-Pro-NO thin film. Next, the Pro-NO template was extracted from MIP-Pro-NO with 0.1 m NaOH. Piezoelectric microgravimetry (PM) on an electrochemical quartz crystal microbalance and electrochemical (differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS)) techniques were used to transduce binding of Pro-NO to molecular cavities of the MIP-Pro-NO. With DPV and EIS chemosensing, the limits of detection (LODs) were about 80.9 and 36.9 nM Pro-NO, respectively; and the selectivity coefficients for urea, glucose, creatinine, and adrenalin interferences were 6.6, 13.2, 2.1, and 2.0, respectively, with DPV as well as 2.3, 2.0, 3.3, and 2.5, respectively, with EIS. With PM under flow injection analysis conditions, the LOD was 10 µm Pro-NO. The MIP-Pro-NO chemosensor detectability and selectivity with respect to interferences were sufficiently high to determine Pro-NO in protein-providing food products.