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. The fluorophore-linked dPxTPs were site-specifically incorporated by PCR into DNA, opposite Ds in templates, with high selectivity. Furthermore, we found that the fluorescence of the triphosphates was partially quenched, but increased upon their incorporation into DNA. These dual fluorophore-quencher base analogues would be useful for site-specific DNA labeling and for monitoring the amplification products of target nucleic acid molecules with a specific sequence. We have demonstrated the utility of the fluorophore-linked Px substrates and the Ds-Px pairing in real-time quantitative PCR for target DNA molecule detection.     

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. Thus, these unnatural Dss-Pn and Dss-Px base pairs function as reporter-quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss-Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss-Pn and Dss-Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore-quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.     

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). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was approximately 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.     

5-Aza-7-deazaguanine–Isoguanine and Guanine–Isoguanine Base Pairs in Watson–Crick DNA: The Impact of Purine Tracts,Clickable Dendritic Side Chains,and Pyrene Adducts

The Watson–Crick coding system depends on the molecular recognition of complementary purine and pyrimidine bases. Now, the construction of hybrid DNAs with Watson–Crick and purine–purine base pairs decorated with dendritic side chains was performed. Oligonucleotides with single and multiple incorporations of 5-aza-7-deaza-2′-deoxyguanosine, its tripropargylamine derivative, and 2′-deoxyisoguanosine were synthesized. Duplex stability decreased if single modified purine–purine base pairs were inserted, but increased if pyrene residues were introduced by click chemistry. A growing number of consecutive 5-aza-7-deazaguanine–isoguanine base pairs led to strong stepwise duplex stabilization, a phenomenon not observed for the guanine–isoguanine base pair. Spacious residues are well accommodated in the large groove of purine–purine DNA tracts. Changes to the global helical structure monitored by circular dichroism spectroscopy show the impact of functionalization to the global double-helix structure. This study explores new areas of molecular recognition realized by purine base pairs that are complementary in hydrogen bonding, but not in size, relative to canonical pairs.     

Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair

Hydrophobic artificial nucleobase pairs without the ability to pair through hydrogen bonds are promising candidates to expand the genetic alphabet. The most successful nucleobase surrogates show little similarity to each other and their natural counterparts. It is thus puzzling how these unnatural molecules are processed by DNA polymerases that have evolved to efficiently work with the natural building blocks. Here, we report structural insight into the insertion of one of the most promising hydrophobic unnatural base pairs, the dDs–dPx pair, into a DNA strand by a DNA polymerase. We solved a crystal structure of KlenTaq DNA polymerase with a modified template/primer duplex bound to the unnatural triphosphate. The ternary complex shows that the artificial pair adopts a planar structure just like a natural nucleobase pair, and identifies features that might hint at the mechanisms accounting for the lower incorporation efficiency observed when processing the unnatural substrates.     

Imidazolo‐dC Metal‐Mediated Base Pairs: Purine Nucleosides Capture Two Ag+ Ions and Form a Duplex with the Stability of a Covalent DNA Cross‐Link

8‐Phenylimidazolo‐dC (^phImidC, 2 ) forms metal‐mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent “purine” 2′‐deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6 . Owing to the ease of nucleobase deprotonation, the new Ag⁺‐mediated base pair containing a “purine” skeleton is much stronger than that derived from the pyrrolo‐ [3,4‐d]pyrimidine system (^phPyrdC, 1 ). The silver‐mediated ^phImidC–^phImidC base pair fits well into the DNA double helix and has the stability of a covalent cross‐link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.     

Unnatural Cytosine Bases Recognized as Thymines by DNA Polymerases by the Formation of the Watson–Crick Geometry

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms by which DNA polymerases faithfully decode chemical information on the template. It was previously shown that two unnatural cytosine bases (termed “M‐fC” and “I‐fC”), which are chemical labeling adducts of the epigenetic base 5‐formylcytosine, can induce C‐to‐T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remains enigmatic. Herein, crystal structures of unnatural cytosine bases pairing to dA/dG in the KlenTaq polymerase‐host–guest complex system and pairing to dATP in the KlenTaq polymerase active site were determined. Both M‐fC and I‐fC base pair with dA/dATP, but not with dG, in a Watson–Crick geometry. This study reveals that the formation of the Watson–Crick geometry, which may be enabled by the A‐rule, is important for the recognition of unnatural cytosines.     

Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues

In an effort to develop an orthogonal third base pair for the storage of genetic information, thiophene and furan heterocycles have been examined as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases. These results expand the potential nucleobase analogues with which the genetic alphabet may be expanded to include five-membered-ring heterocycles.     

Synthesis,Molecular Recognition,and Enzymology of Oligonucleotides Containing the Non-standard Base Pair between 5-Aza-7-deazaisoguanine and 6-Amino-3-methylpyrazin-2(1H)-one,a Donor-Acceptor-Acceptor Purine Analog and an Acceptor-Donor-Donor Pyrimidine Analog

A 6-aminopyrazin-2(1H)-one (pyADD), when incorporated as a pyrimidine-base analog into an oligonucleotide chain, presents a H-bond acceptor-donor-donor pattern to 5-aza-7-deazaisoguanine (puDAA), the complementrary donor-acceptor-acceptor purine analog. Reported here are the syntheses of the phosphoramidite of the 2′-deoxyribonucleoside bearing the puDAA base, oligonucleotides containing this nucleoside unit, the enzyme-assisted synthesis of oligoribonucleotides containing the pyADD ribonucleoside, and the molecular-recognition properties of this non-standard base pair in an oligonucleotide context. A series of melting experiments suggests that the pyADD · puDAA base pair contributes to the relative stability of a duplex structure approximately the same as an A · T base pair, and significantly more than mismatches between these non-standard bases and certain standard nucleobases. The pyADD nucleoside bisphosphate is accepted by T4 RNA ligase, but the triphosphate of the pyADD nucleoside was not incorported by T7 RNA polymerase opposite the puDAA nucleobase in a template. Oligonucleotides containing the pyADD base slowly undergo a reversible first-order reaction, presumably an epimerization process to give the α-D -anomer. These experiments provide the tools for laboratory-based use of the pyADD · puDAA base pair as a component of an oligonucleotide-like molecular-recognition system based on an expanded genetic alphabet.     

Solution Structure,Mechanism of Replication,and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge‐on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue d MMO2 , which forms an unnatural base pair with the nucleotide analogue d 5SICS . Replacing the para‐methyl substituent of d MMO2 with an annulated furan ring (yielding d FMO ) has a dramatically negative effect on replication, while replacing it with a methoxy (d DMO ) or with a thiomethyl group (d TMO ) improves replication in both steady‐state assays and during PCR amplification. Thus, d TMO –d 5SICS , and especially d DMO –d 5SICS , represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental d MMO2 –d 5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.     

Hydration and stability of nucleic acid bases and base pairs

Empirical, quantum chemical calculations and molecular dynamics simulations of the role of a solvent on tautomerism of nucleic acid bases and structure and properties of nucleic acid base pairs are summarized. Attention was paid to microhydrated (by one and two water molecules) complexes, for which structures found by scanning of empirical potential surfaces were recalculated at a correlated ab initio level. Additionally, isolated as well as mono- and dihydrated H-bonded, T-shaped and stacked structures of all possible nucleic acid base pairs were studied at the same theoretical levels. We demonstrate the strong influence of a solvent on the tautomeric equilibrium between the tautomers of bases and on the spatial arrangement of the bases in a base pair. The results provide clear evidence that the prevalence of either the stacked or hydrogen-bonded structures of the base pairs in the solvent is not determined only by its bulk properties, but rather by specific hydrophilic interactions of the base pair with a small number of solvent molecules.     

A biosynthetic route to photoclick chemistry on proteins

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.