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Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

A new pair of unnatural base pair is developed between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which has functioned in DNA amplification. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair can be used for expanded genetic systems in DNA-based biotechnology.

Site-specific fluorescent labeling of RNA molecules is achieved by specific transcription using an unnatural base pair system. This method is useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.

A two-unnatural-base-pair system is developed for coupled transcription-translation systems with the expanded genetic code. The unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.

Gel mobility and dna-specific exonuclease resistance analyses were used to investigate the ability of gna trinucleotide loop fragments d(GCGAAAGC) and d(GCGAAGC) in producing stable dna hairpins with Tm values of 76 degrees Celsius. The formed minihairpins exhibited high mobility both on polycramide and 7 M urea combined gel and resistance to T4 dna polymerase activity denoting extraordinary stability. Structure and nature of four fragments, fragments d(GCGNAC) (N=A,G,C, and T), were analyzed using NMR and thermodynamic studies which revealed folded structure believed to be advantageous for minihairpin formation.

A comparaison of the inhibitory activity of NH2-terminal human cysteine proteinase fused with porcine adenylate kinase shows that the amino-terminal is necessary for its activity. Removal of six residues from the amino-terminal decreases the inhibitory activity while removal of 15 residues completely inactivates the inhibitor. However the removal of the Met residue from the amino-terminal or substitution of the Pro3 with Leu residue does not affect the inhibitory activity. Substitution of the Gky75 with His residue decreases the loss of activity.