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Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors < 10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a ~800 nucleotide RNA in which every cytosine was replaced with tC. 10.1021/ac902456n

Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA--protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5'triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase, tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long [IV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies. 10.1021/ac9017555

A fluorescence resonance energy transfer (FRET) system is reported which monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. The results indicate that FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.

In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.

A method for the accurate determination of the fraction of surface-attached DNA duplexes exhibiting a single-fluorophore-labeled nucleobase is introduced. The fluorescence signals obtained from surface plasmon field-enhanced fluorescence spectroscopy along with the optical properties of the sensor architecture determined by surface plasmon resonance were employed for the calculation. A Cy5-labeled nucleotide was incorporated into DNA at a well-defined position via template-directed DNA synthesis performed by a DNA polymerase. The sample-to-sample variations associated with the optical properties of the employed metal films caused a small variation in the strength of the evanescent field. This variation was accounted for by evanescent field integration over the DNA layers. The exponential-type relationship between the fraction of DNA with Cy5-dCTP incorporation at the surface and the mole fraction of the Cy5-dCTP in solution indicates the preferential incorporation of nonlabeled nucleotides by the DNA polymerase.

The studies related to the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase [alpha] and Klenow fragment of DNA polymerase I (Escherichia coli) are described. Both enzymes showed the aptitude to readily extend the formed tC(o)-G and tC(o)-A base pairs and are able to incorporate at least four consecutive nucleotide analogues.

A new concept for the fusion of programmable phospholipid vesicles, based on the hybridization of membrane-anchored DNA strands is being described. The fusion is found to be dependent on the strength of the membrane anchor, as well as the length of the DNA.