Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet

DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogues, we performed a detailed structure-activity relationship analysis, which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.     

Hg(II) ion specifically binds with T:T mismatched base pair in duplex DNA

Metal-mediated base pair formation, resulting from the interaction between metal ions and artificial bases in oligonucleotides, has been developed for its potential application in nanotechnology. We have recently found that the T:T mismatched base pair binds with Hg(II) ions to generate a novel metal-mediated base pair in duplex DNA. The thermal stability of the duplex with the T-Hg-T base pair was comparable to that of the corresponding T:A or A:T. The novel T-Hg-T base pair involving the natural base thymine is more convenient than the metal-mediated base pairs involving artificial bases due to the lack of time-consuming synthesis. Here, we examine the specificity and thermodynamic properties of the binding between Hg(II) ions and the T:T mismatched base pair. Only the melting temperature of the duplex with T:T and not of the perfectly matched or other mismatched base pairs was found to specifically increase in the presence of Hg(II) ions. Hg(II) specifically bound with the T:T mismatched base pair at a molar ratio of 1:1 with a binding constant of 10(6) M(-1), which is significantly higher than that for nonspecific metal ion-DNA interactions. Furthermore, the higher-order structure of the duplex was not significantly distorted by the Hg(II) ion binding. Our results support the idea that the T-Hg-T base pair could eventually lead to progress in potential applications of metal-mediated base pairs in nanotechnology.     

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.     

Cyclohexyl "base pairs" stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.     

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.     

Optimization of interstrand hydrophobic packing interactions within unnatural DNA base pairs

As part of an effort to expand the genetic alphabet, we have evaluated a large number of predominantly hydrophobic unnatural base pairs. We now report the synthesis and stability of unnatural base pairs formed between simple phenyl rings modified at different positions with methyl groups. Surprisingly, several of the unnatural base pairs are virtually as stable as a natural base pair in the same sequence context. The results show that neither hydrogen-bonding nor large aromatic surface area are required for base pair stability within duplex DNA and that interstrand interactions between small aromatic rings may be optimized for both stability and selectivity. These smaller nucleobases are not expected to induce the distortions in duplex DNA or at the primer terminus that seem to limit replication of larger unnatural base pairs, and they therefore represent a promising approach to the expansion of the genetic alphabet.     

Calculation of the stabilization energies of oxidatively damaged guanine base pairs with guanine

DNA is constantly exposed to endogenous and exogenous oxidative stresses. Damaged DNA can cause mutations, which may increase the risk of developing cancer and other diseases. G:C-C:G transversions are caused by various oxidative stresses. 2,2,4-Triamino-5(2H)-oxazolone (Oz), guanidinohydantoin (Gh)/iminoallantoin (Ia) and spiro-imino-dihydantoin (Sp) are known products of oxidative guanine damage. These damaged bases can base pair with guanine and cause G:C-C:G transversions. In this study, the stabilization energies of these bases paired with guanine were calculated in vacuo and in water. The calculated stabilization energies of the Ia:G base pairs were similar to that of the native C:G base pair, and both bases pairs have three hydrogen bonds. By contrast, the calculated stabilization energies of Gh:G, which form two hydrogen bonds, were lower than the Ia:G base pairs, suggesting that the stabilization energy depends on the number of hydrogen bonds. In addition, the Sp:G base pairs were less stable than the Ia:G base pairs. Furthermore, calculations showed that the Oz:G base pairs were less stable than the Ia:G, Gh:G and Sp:G base pairs, even though experimental results showed that incorporation of guanine opposite Oz is more efficient than that opposite Gh/Ia and Sp.     

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like C?Silver(I)?C Metallo‐Base Pairs

Metallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T Hg^II T base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases C Ag^I C formed in an RNA duplex. One Ag^I occupies the center between two cytosines and forms a C Ag^I C base pair through N3 Ag^I N3 linear coordination. The C Ag^I C base pair formation does not disturb the standard A‐form conformation of RNA. Since the C Ag^I C base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.     

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T? Hg^II? T base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases C? Ag^I? C formed in an RNA duplex. One Ag^I occupies the center between two cytosines and forms a C? Ag^I? C base pair through N3? Ag^I? N3 linear coordination. The C? Ag^I? C base pair formation does not disturb the standard A‐form conformation of RNA. Since the C? Ag^I? C base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.     

碱基对在DNA双螺旋链上分离的自由能计算

DNA是大部分生物包括病毒的基因载体。DNA双螺旋链通过A＝T和G≡C两种碱基对编码实现对遗传信息的存储。碱基对中的相互作用对DNA双螺旋链的稳定性起到重要作用,直接关系到基因的复制和转录。当前研究中,我们构建了四组不同结构的DNA双螺旋链,进行了总共4.3 μs的分子动力学模拟。通过伞形取样技术计算了DNA双螺旋链中碱基对分离的自由能曲线,并从分子尺度细节和相互作用能对自由能曲线进行解析。在碱基对G≡C的自由能曲线(PMF-PGC)上观察到三个峰,通过监测氢键数目的变化发现分别对应于G≡C三个氢键的断裂;而在A＝T的自由能曲线(PMF-PAT)上只出现一个峰,说明A＝T的两个氢键在分离过程中几乎同时断裂。PMF-PGC的总能垒比PMF-PAT高,主要是因为G≡C比A＝T多一个氢键,更稳定。两条曲线的后段自由能仍然升高,而此时碱基对的氢键已断裂,这是DNA链骨架刚性所导致。我们还研究了碱基对稳定性受相邻碱基对的影响,发现邻近G≡C碱基对会增强A＝T的稳定性, C≡G会削弱A＝T的稳定性, T＝A对A＝T的影响较小。     

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.     

Optimization of unnatural base pair packing for polymerase recognition

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.     

Solution structure of S-DNA formed by covalent base pairing involving a disulfide bond

Here, we present the solution structure of a DNA duplex containing a disulfide base pair (S-DNA). The unnatural nucleoside "S" possessing a thiophenyl group as base was incorporated into a self-complementary singled-stranded oligonucleotide. Crosslinking of the disulfide base pair was analyzed by non-denaturing polyacrylamide gel electrophoresis. Under oxidizing conditions a high molecular weight band as 18 mer, corresponding to the double-stranded molecule (5'-GCGASTCGC: 3'-CGCTSAGCG), was found, whereas single-stranded self-complementary 9 mer oligonucleotide GCGASTCGC was detected in the presence of a reducing agent. These results suggest that the oligonucleotide is covalently linked by disulfide bonding under oxidizing conditions, which can be reversibly reduced to two thiol groups under reducing conditions. CD spectrum of S-DNA (CGASTCG) under oxidizing conditions suggested that the duplex had a right-handed double-stranded structure similar to that of natural DNA (B-form, CGATCG). NMR studies confirmed that this CGASTCG resembled natural B-DNA and that the two phenyl rings derived from the disulfide base pairing intercalated into the duplex. However, these two phenyl rings were not positioned in the same plane as the other base pairs. Specifically, NOEs suggest that although CGASTCG adopts a structure similar to B-type DNA, the S-DNA duplex is bent at the point of disulfide base pairing to face the major groove.     

Factors determining the deriving force of DNA formation: geometrical differences of base pairs, dehydration of bases, and the arginine assisting

The mechanism of the fidelity synthesis of DNA associated with the process of dGTP combination to the DNA template was explored. The exclusion of water molecules from the hydrated DNA bases can amplify the energy difference between the correct and incorrect base pairs, but the effect of the water molecules on the Gibbs free energy of formation is dependent on the binding sites for the water molecules. The water detachment from the incoming dNTP is not the only factor but the first step for the successful replication of DNA. The second step is the selection of the DNA polymerase on the DNA base pair through the comparison between the correct DNA base and the incorrect DNA base. The bonding of the Arg668 with the incoming dNTP can enlarge the Gibbs free energies of formation of the base pairs, especially the correct base pairs, thus increasing the driving force of DNA formation. When the DNA base of the primer terminus is correct, the extension of the guanine and the adenine is quicker than that of the cytosine and the thymine because of the hydrogen bonding fork formation of Arg668 with the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP. Because of the geometry differences of the incorrect base pairs with the correct base pairs, the effect from the DNA polymerase is smaller on the incorrect base pair than on the correct base pair, and the extension of a mispair is slower than that of a correct base pair. This decreases the extension rate of the base pair and thus allows proofreading exonuclease activity to excise the incorrect base pair. Arg668 cannot prevent the extension of the GT mispair, as well as the GC correct base pair, and GA and GG mispairs. This may be attributed to the small geometry difference between the GT base pair and the correct AT base pair.     

Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs

Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.     

Hetero‐Selective DNA‐Like Duplex Stabilized by Donor–Acceptor Interactions

We report on the characterization of a novel hetero‐selective DNA‐like duplex of pyrene and anthraquinone pseudo base pairs. The pyrene/anthraquinone pairs showed excellent selectivity in hetero‐recognition and even trimers were found to form a hetero‐duplex. Pyrene and anthraquinone moieties were tethered on acyclic D ‐threoninol linkers and linked to adjacent residues by using standard phosphoramidite chemistry. When pyrene and anthraquinone were incorporated at pairing positions in complementary strands of natural DNA oligonucleotides, the duplex was stabilized significantly. Moreover, a pyrene hexamer and an anthraquinone hexamer formed a stable artificial hetero‐duplex without the assistance of natural base pairs. The pyrene/anthraquinone pair was so stable that even trimers formed a hetero‐duplex under conditions in which natural DNA strands of three residues do not.     

Hydrophobic, Non-Hydrogen-Bonding Bases and Base Pairs in DNA

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.     

Electronic properties of metal-modified DNA base pairs

The electronic properties of several metal-modified Watson-Crick guanine-cytosine base pairs are investigated by means of first-principle density functional theory calculations. Focus is placed on a new structure recently proposed as a plausible model for building an antiparallel duplex with Zn-guanine-cytosine pairs, but we also inspect several other conformations and the incorporation of Ag and Cu ions. We analyze the effects induced by the incorporation of one metal cation per base pair by comparing the structures and the electronic properties of the metalated pairs to those of the natural guanine-cytosine pair, particularly for what concerns the modifications of energy levels and charge density distributions of the frontier orbitals. Our results reveal the establishment of covalent bonding between the metal cation and the nucleobases, identified in the presence of hybrid metal-guanine and metal-cytosine orbitals. Attachment of the cation can occur either at the N1 or the N7 site of guanine and is compatible with altering or not altering the H-bond pattern of the natural pair. Cu(II) strongly contributes to the hybridization of the orbitals around the band gap, whereas Ag(I) and Zn(II) give hybrid states farther from the band gap. Most metalated pairs have smaller band gaps than the natural guanine-cytosine pair. The band gap shrinking along with the metal-base coupling suggests interesting consequences for electron transfer through DNA double helices.     

Vibrational dynamics of DNA. I. Vibrational basis modes and couplings

Carrying out density functional theory calculations of four DNA bases, base derivatives, Watson-Crick (WC) base pairs, and multiple-layer base pair stacks, we studied vibrational dynamics of delocalized modes with frequency ranging from 1400 to 1800 cm(-1). These modes have been found to be highly sensitive to structure fluctuation and base pair conformation of DNA. By identifying eight fundamental basis modes, it is shown that the normal modes of base pairs and multilayer base pair stacks can be described by linear combinations of these vibrational basis modes. By using the Hessian matrix reconstruction method, vibrational coupling constants between the basis modes are determined for WC base pairs and multilayer systems and are found to be most strongly affected by the hydrogen bonding interaction between bases. It is also found that the propeller twist and buckle motions do not strongly affect vibrational couplings and basis mode frequencies. Numerically simulated IR spectra of guanine-cytosine and adenine-thymine bases pairs as well as of multilayer base pair stacks are presented and described in terms of coupled basis modes. It turns out that, due to the small interlayer base-base vibrational interactions, the IR absorption spectrum of multilayer base pair system does not strongly depend on the number of base pairs.     

The elusive atomic rationale for DNA base pair stability

A systematic analysis of the electrostatic interaction between 27 natural DNA base pairs was carried out, based on ab initio correlated wave functions and the topology of the electron density. Using high rank multipole moments we show that the atomic partitioning of the interaction energy contains many substantial contributions between distant atoms. Profiles of cumulative energy versus internuclear distance show large fluctuations and provide an electrostatic fingerprint of the partitioning of interaction energy in a complex. A quantified comparison between each pair of energy profiles, one for each base pair, makes clear that there is no correlation between the total base pair interaction energy and the shape of the profile. In other words, base pairs with similar interaction energy are not stable for the same reasons in terms of atomic partitioning. In summary, simple rules to rationalize the pattern of energetic stability of naturally occurring base pairs in terms of subsets of atoms are elusive. Our work cautions against inappropriate use of Jorgensen's secondary interaction hypothesis.