Highly specific unnatural base pair systems as a third base pair for PCR amplification.

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity.

Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system.

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification.

A new unnatural base pair system between fluorophore and quencher base analogues for nucleic acid-based imaging technology.

In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH(2)-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2'-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent.

A unique fluorescent base analogue for the expansion of the genetic alphabet.

Fluorescent nucleobase analogues are useful in a wide variety of biology and biotechnology tools as molecular probes and reporters for nucleic acids. Here we present a novel fluorescent purine analogue, 7-(2,2'-bithien-5-yl)-imidazo[4,5-b]pyridine (denoted as Dss). The nucleoside triphosphates of Dss can be site-specifically incorporated into DNA and RNA by polymerases, opposite its pairing partner, pyrrole-2-carbaldehyde (Pa), in DNA templates.

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules.

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3'-->5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base.

Efficient PCR amplification by an unnatural base pair system.

Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR.

Sequences around the unnatural base pair in DNA templates for efficient replication.

Unnatural base pairs, compatible with PCR amplification, could potentially increase the versatility of nucleic acids. We recently reported an unnatural base pair, between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in PCR. Toward the efficient incorporation of extra, functional components into DNA fragments, we examined the influence of the sequences around the unnatural base pair and the dependence of the substrate concentrations on the selectivity and efficiency of replication by DNA polymerase.

Development of an unnatural base pair for efficient PCR amplification.

An unnatural base pair system could expand the genetic alphabet, enabling the site-specific incorporation of extra, functional components into nucleic acids and proteins. We developed an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in DNA amplification by PCR. After 20 cycles of PCR, the mutation rate of the Ds-Pn pair in an amplified DNA fragment was approximately 1%.

An efficient unnatural base pair for PCR amplification.

Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa).

Fluorescent probing for RNA molecules by an unnatural base-pair system.

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates.

An unnatural base pair system for in vitro replication and transcription.

The development of unnatural base pairs that function in replication, transcription, and translation could expand the genetic alphabet and enable the site-specific incorporation of functional components into nucleic acids and proteins. We present an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and pyrrole-2-carbaldehyde (denoted by Pa). In replication, the Ds-Pa pair exhibits high selectivity in combination with the usual and modified triphosphate substrates and exonuclease-proficient DNA polymerases.

Site-specific incorporation of fluorescent probes into RNA by specific transcription using unnatural base pairs.

To analyze the local conformational changes of RNA molecules, we developed a site-specific fluorescent labeling method for RNA fragments by T7 transcription, using unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y) and between 2-amino-6-(2-thiazolyl)purine (v) and y. Ribonucleoside 5'-triphosphates of 5-fluorescence-linked y derivatives can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Using this specific transcription, the substrate of a fluorescein-linked y was introduced into a theophylline-binding RNA aptamer.

Fluorescent properties of an unnatural nucleobase, 2-amino-6-(2-thienyl)purine, in DNA and RNA fragments.

Nucleoside derivatives of 2-amino-6-(2-thienyl)purine (s) are fluorescent and can be site-specifically incorporated into RNA by transcription mediated by unnatural base pairs between s and its complementary bases. To utilize the fluorescent s base as a probe, we examined the fluorescent properties of s in DNA and RNA fragments. The nucleoside of s exhibited a fluorescence emission centered at 432 nm, characterized by two major excitation maxima (299 and 352 nm), and its quantum yield was 0.

Non-hydrogen-bonded base pairs for specific transcription.

Specific transcription mediated by unnatural base pairs could create novel RNA molecules with increased functionality and expand the genetic code. Here, we report an unnatural base pair between pyrrole-2-carbaldehyde (Pa) and 2-amino-6-(2-thienyl)purine (s) or 6-(2-thienyl)purine (s') for the site-specific incorporation of s or s' into RNA by T7 RNA polymerase, using DNA templates containing Pa. Despite the absence of significant hydrogen bonding interactions between the unnatural bases, the efficiency and fidelity of the s-Pa pairing in transcription were as high as those of the natural base pairings.

Site-specific fluorescent labeling of RNA by a base-pair expanded transcription system.

Ribonucleoside 5'-triphosphates of fluorescently-labeled 2-oxo(1H)pyridines, in which carboxyfluorescein (FAM) or carboxytetramethylrhodamine (TAMRA) was attached at position 5, were chemically synthesized. These substrates were site-specifically incorporated into RNA by T7 transcription mediated by unnatural base pairs between 2-amino-6-(2-thienyl)purine and 2-oxo(1H)pyridine or 2-amino-6-(2-thiazolyl)purine and 2-oxo(1H)pyridine. This site-specific fluorescent labeling of RNA fragments will be a powerful tool for the fields of nucleic acid chemistry, biology, and technology.

An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA.

Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions.

Site-specific fluorescent labeling of RNA molecules by specific transcription using unnatural base pairs.

Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine (v) and y function in transcription, and the substrates of y and 5-modified y bases can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Ribonucleoside 5'-triphosphates of 5-fluorophore-linked y bases were chemically synthesized from the nucleoside of y.

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs.

Direct site-specific biotinylation of RNA molecules was achieved by specific transcription mediated by unnatural base pairs. Unnatural base pairs between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y), or 2-amino-6-(2-thiazolyl)purine (denoted as v) and y specifically function in T7 transcription. Using these unnatural base pairs, the substrate of biotinylated-y (Bio-yTP) was selectively incorporated into RNA, opposite s or v in the DNA templates, by T7 RNA polymerase.

An efficient unnatural base pair for a base-pair-expanded transcription system.

For the site-specific incorporation of artificial components into RNA by transcription, an efficient, unnatural base pair between 2-amino-6-(2-thiazolyl)purine (denoted as v) and 2-oxo(1H)pyridine (denoted as y) was developed. The substrates of y and 5-substituted y were site-specifically incorporated into RNA by T7 RNA polymerase opposite v in templates. The efficiency and fidelity of the v-y pairing in transcription were as high as those of the natural A-T(U) and G-C pairings.

A two-unnatural-base-pair system toward the expansion of the genetic code.

Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription.

Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs.

A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (Iy) capable of site-specific incorporation into transcripts was developed. The site-specific Iy incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between Iy and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, Iy was incorporated into an anti(Raf-1) RNA aptamer, which binds to human Raf-1 and inhibits the interaction between Raf-1 and Ras.

Unnatural base pairs mediate the site-specific incorporation of an unnatural hydrophobic component into RNA transcripts.

Site-specific incorporation of a hydrophobic nucleotide analog into RNA, by T7 transcription mediated by unnatural base pairs, was developed. The nucleotide analog, 5-phenylethynyl-3-(beta-D-ribofuranosyl)pyridin-2-one 5-triphosphate (denoted by Ph-yTP), was chemically synthesized and then site-specifically incorporated by T7 RNA polymerase into RNA opposite the pairing partner, 2-amino-6-(2-thienyl)purine (denoted by s) in DNA templates. The introduction of Ph-y into a theophylline-binding RNA aptamer, in which a uridine in the internal loop was replaced by Ph-y, raised the thermal stability of the aptamer.

An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine.

To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as Pa') and synthesized the nucleoside derivatives of Pa'. The base pairing of Pa' with the partner, 9-methylimidazo[(4,5)-b]pyridine (Q), was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q. The thermal stability of a DNA duplex containing the Q-Pa' pair and the incorporation efficiency of the Pa' substrate (dPa'TP) into DNA opposite Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q-Pa pair.

Simple preparation of biotinylated RNA by transcription via an unnatural base pair.

To develop a convenient method for RNA fragment biotinylation at a specific position, we synthesized ribonucleoside 5'-triphosphates of biotinylated unnatural bases. These unnatural bases were composed of 2-oxo(1H)pyridine (denoted as y), specifically pairing with 2-amino-6-(2-thienyl)purine (denoted as s) in transcription. The y base was linked with biotin through an allylamine or propynylamine linker at position 5 of y.

An unnatural base pair for efficient incorporation of nucleotide analogs into RNAs.

An unnatural base, 2-amino-6-(2-thiazolyl)purine (denoted as v), was developed as an efficient complementary base of another unnatural base, 2-oxo(1H)pyridine (denoted as y). The nucleoside derivatives of v and DNA fragments containing v were chemically synthesized. The efficiency and selectivity of the v-y pairing in replication and transcription were examined and compared to those of a previously developed unnatural base pair between 2-amino-6-(2-thienyl)purine (s) and y.