Abstract
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-to‑phosphorus 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.
Highlights
A biopolymer with precisely positioned monomer units in its structure may reveal unique properties useful for molecular recognition, biocatalysis, and molecular encoding of hereditary information [1,2]
Interactions of the molecularly imprinted polymer (MIP) film with amino acids were examined by Piezoelectric microgravimetry (PM) under flow injection analysis (FIA) conditions with water serving as the carrier liquid
With X-ray photoelectron spectroscopy (XPS) depth profiling, we demonstrated a strong relationship between nucleobase density in the MIP and, MIP nucleobase affinity to their cognate amino acids
Summary
A biopolymer with precisely positioned monomer units (or functionalities) in its structure may reveal unique properties useful for molecular recognition, biocatalysis, and molecular encoding of hereditary information [1,2]. DNA-directed electropolymerization of functional monomers around the ODN template resulted in nucleobase sequence controlling at the molecular level in cavities of the MIP [8,9]. The TATA box can be considered as a small isolated fragment of a whole DNA damage repairing system Over the years, both direct and indirect sequence-specific DNA recognition by proteins has been identified [21,22,23]. As a proof of concept, we performed that way the DNA-directed electropolymerization resulting in the artificial singlestranded TTTATA oligonucleotide This oligonucleotide hybridized with the complementary 5′-TATAAA-3′ template to form a double-stranded hybrid in the MIP. It appears that amino acids that recognize the natural TATA box fairly well recognize our artificial TATA box
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