Two are not enough: synthetic strategies and applications of unnatural base pairs.

Nucleic acid chemistry is a rapidly evolving field, and the need for novel nucleotide modifications and artificial nucleotide building blocks for diagnostic and therapeutic use, material science or for studying cellular processes continues unabated. This review focusses on the development and application of unnatural base pairs as part of an expanded genetic alphabet. Not only recent developments in "nature-like" artificial base pairs are presented, but also current synthetic methods to get access to C-glycosidic nucleotides. Wide-ranging viability in synthesis is a prerequisite for the successful use of unnatural base pairs in a broader spectrum and will be discussed.

Structural Properties of Hachimoji Nucleic Acids and Their Building Blocks: Comparison of Genetic Systems with Four, Six and Eight Alphabets.

Expansion of the genetic alphabet is an ambitious goal. A recent breakthrough has led to the eight-base (hachimoji) genetics having canonical and unnatural bases. However, very little is known on the molecular-level features that facilitate the candidature of unnatural bases as genetic alphabets. Here we amalgamated DFT calculations and MD simulations to analyse the properties of the constituents of hachimoji DNA and RNA. DFT reveals the dominant syn conformation for isolated unnatural deoxyribonucleosides and at the 5'-end of oligonucleotides, although an anti/syn mixture is predicted at the nonterminal and 3'-terminal positions. However, isolated ribonucleotides prefer an anti/syn mixture, but mostly prefer anti conformation at the nonterminal positions. Further, the canonical base pairing combinations reveals significant strength, which may facilitate replication of hachimoji DNA. We also identify noncanonical base pairs that can better tolerate the substitution of unnatural pairs in RNA. Stacking strengths of 51 dimers reveals higher average stacking stabilization of dimers of hachimoji bases than canonical bases, which provides clues for choosing energetically stable sequences. A total of 14.4 µs MD simulations reveal the influence of solvent on the properties of hachimoji oligonucleotides and point to the likely fidelity of replication of hachimoji DNA. Our results pinpoint the features that explain the experimentally observed stability of hachimoji DNA.

Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity.

The high fidelity poses a central role in developing unnatural base pairs (UBPs), which means the high pairing capacity of unnatural bases with their partners, and low mispairing with all the natural bases. Different strategies have been used to develop higher-fidelity UBPs, including optimizing hydrophobic interaction forces between UBPs. Variant substituent groups are allowed to fine tune the hydrophobic forces of different UBPs' candidates. However, the modifications on the skeleton of TPT3 base are rare and the replication fidelity of TPT3-NaM remains hardly to improve so far. In this paper, we reasoned that modifying and/or expanding the aromatic surface by Bromo-substituents to slightly increase hydrophobicity of TPT3 might offer a way to increase the fidelity of this pair. Based on the hypothesis, we synthesized the bromine substituted TPT3, 2-bromo-TPT3 and 2, 4-dibromo-TPT3 as the new TPT3 analogs. While the enzyme reaction kinetic experiments showed that d2-bromo-TPT3-dNaM pair and d2, 4-dibromo-TPT3TP-dNaM pair had slightly less efficient incorporation and extension rates than that of dTPT3-dNaM pair, the assays did reveal that the mispairing of 2-bromo-TPT3 and 2, 4-dibromo-TPT3 with all the natural bases could dramatically decrease in contrast to TPT3. Their lower mispairing capacity promoted us to run polymerase chain amplification reactions, and a higher fidelity of d2-bromo-TPT3-dNaM pair could be obtained with 99.72 ± 0.01% of the in vitro replication fidelity than that of dTPT3-dNaM pair, 99.52 ± 0.09%. In addition, d2-bromo-TPT3-dNaM can also be effectively copied in E. coli cells, which showed the same replication fidelity as that of dTPT3-dNaM in the specific sequence, but a higher fidelity in the random sequence context.

Enzymatic Synthesis of the Unnatural Nucleotide 2'-Deoxyisoguanosine 5'-Monophosphate.

Naturally occurring DNA contains four canonical bases, forming two Watson-Crick base pairs (adenine-thymine, guanine-cytosine). Efforts over the past decades have led to the development of several unnatural base pairs, enabling the synthesis of unnatural DNA with an expanded genetic alphabet. The engineering of organisms capable of de novo biosynthesis of unnatural DNA would have significant technological and philosophical implications, but remains a challenge. Here we report the enzymatic conversion of 2'-deoxyxanthosine 5'-monophosphate (dXMP) into deoxyisoguanosine monophosphate (dBMP), a precursor of the unnatural isoguanine-isocytosine base pair. The reaction is catalyzed by the bacteriophage enzyme PurZ and bacterial PurB, and is a key addition to the toolbox for de novo biosynthesis of unnatural DNA.

Mechanistic Insight into the Photoinduced Damage of an Unnatural Base Pair.

Chemical- and photostability of unnatural base pairs (UBPs) are important to maintain the genetic code integrity, and critical for developing healthy semisynthetic organisms. As reported, dTPT3 was less stable upon irradiation, and thus might act as a pervasive photosensitizer to induce oxidative damage within DNA, causing harm to living semi-synthetic organisms when exposed to UVA radiation. However, there was no knowledge about molecular-level understanding of this damage process. In this paper, we not only identified four photoproducts of dTPT3, including desulfur-dTPT3 (dTPT3H ), TPT3 sulphinate (TPT3SO2 ), TPT3 sulphonate (TPT3SO3 ) and TPT3-thioTPT3 (TPT3S TPT3), but also established a Type II photosensitized oxidation mechanism. In addition, the antioxidant (sodium ascorbate) was able to effectively inhibit the photoproducts formation of dTPT3 and dTPT3 in DNA, suggesting that a reductive environment might protect DNA bearing dTPT3 against UVA oxidation and ameliorate its adverse biological effects. The comprehensive understanding of TPT3' photochemical stability will give researchers helpful guidance to design more photostable UBPs and construct healthier semisynthetic organisms.

Dye-Conjugated Spinach RNA by Genetic Alphabet Expansion.

Light-emitting systems using an RNA aptamer-dye pair, such as Spinach RNA, are an attractive method for imaging and tracing RNA expression in vitro and in vivo. We present an alternative Spinach method by genetic alphabet expansion using an unnatural base pair system, in which a dye-conjugated unnatural base substrate is site-specifically incorporated at a specific position in Spinach RNA by transcription involving the third base pair. The incorporation position was predicted by molecular dynamics simulations. This dye-conjugated Spinach RNA increased the thermal stability of the fluorescence, the robustness against ion sensitivity, and the resistance against photobleaching. Furthermore, we applied our method to Baby Spinach, a shorter version of Spinach, for dye conjugation toward the visible detection of transcripts. This is the first demonstration of an alternative RNA imaging method for a detection system using genetic alphabet expansion.

Hydrogen Bonding in Natural and Unnatural Base Pairs-A Local Vibrational Mode Study.

In this work hydrogen bonding in a diverse set of 36 unnatural and the three natural Watson Crick base pairs adenine (A)-thymine (T), adenine (A)-uracil (U) and guanine (G)-cytosine (C) was assessed utilizing local vibrational force constants derived from the local mode analysis, originally introduced by Konkoli and Cremer as a unique bond strength measure based on vibrational spectroscopy. The local mode analysis was complemented by the topological analysis of the electronic density and the natural bond orbital analysis. The most interesting findings of our study are that (i) hydrogen bonding in Watson Crick base pairs is not exceptionally strong and (ii) the N-H⋯N is the most favorable hydrogen bond in both unnatural and natural base pairs while O-H⋯N/O bonds are the less favorable in unnatural base pairs and not found at all in natural base pairs. In addition, the important role of non-classical C-H⋯N/O bonds for the stabilization of base pairs was revealed, especially the role of C-H⋯O bonds in Watson Crick base pairs. Hydrogen bonding in Watson Crick base pairs modeled in the DNA via a QM/MM approach showed that the DNA environment increases the strength of the central N-H⋯N bond and the C-H⋯O bonds, and at the same time decreases the strength of the N-H⋯O bond. However, the general trends observed in the gas phase calculations remain unchanged. The new methodology presented and tested in this work provides the bioengineering community with an efficient design tool to assess and predict the type and strength of hydrogen bonding in artificial base pairs.

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA.

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.

The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases.

Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen-bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base-pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.

EPR Distance Measurements on Long Non-coding RNAs Empowered by Genetic Alphabet Expansion Transcription.

We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3NO and its application for site-specific spin-labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin-labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter-spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self-complementary RNA sequences and in a well-studied 185 nucleotide non-coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A-region of the long non-coding RNA Xist, by PELDOR.

Towards Reverse Transcription with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX). However, the application of such UBPs in reverse transcription (rtc), which is a key step for RNA-based SELEX, has not been reported so far. The implication of Romesberg's NaM:TPT3 base pair in rtc reactions is presented by testing five commercially available reverse transcriptases (RTs). The employed RTs predominantly pause at the site of the unnatural nucleotide rTPT3 not being able to accept the dNaM building block as a substrate. This allows verification of the unnatural base position in RNA and an estimation of their abundance. In contrast, primer extension from an rNaM-containing template results in considerably more full-length cDNA. Furthermore, RTs that could potentially be able to handle an expanded genetic alphabet based on NaM:TPT3 are presented.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

Structure and Electronic Properties of Unnatural Base Pairs: The Role of Dispersion Interactions.

Recent reports of the successful incorporation of unnatural base pairs (UBPs), such as d5SICS-dNaM, in the gene sequence and replication with DNA is an important milestone in synthetic biology. Followed by this, several other UBPs, such as dTPT3-dNaM, dTPT3-dFIMO, dTPT3-IMO, dTPT3-FEMO, FTPT3-NaM, FTPT3-FIMO, FTPT3-IMO, and FTPT3-FEMO, have demonstrated similar or better retention and fidelity inside cells. Of these base pairs, dNaM-dTPT3 has been optimized to be a better fit inside a pAIO plasmid. Based on both implicit and explicit dispersion-corrected density functional theory (DFT) calculations, we show that although this set of UBPs is significantly diverse in elemental and structural configuration, the members do share a common trait of favoring a slipped parallel stacked dimer arrangement. Unlike the natural bases (A, T, G, C, and U), this set of UBPs has a negligible affinity for a Watson-Crick (WC)-type planar structure because they are invariably more stable within slipped parallel stacked orientations. We also observed that all the UBPs have either similar or higher binding energies with the natural bases in similar stacked orientations. When arranged between two natural base pairs, the UBPs exhibited a binding energy similar to that of three-base sequences of natural bases. Our computational data show that the most promising base pairs are 5SICS-NaM, TPT3-NaM, and TPT3-FEMO. These results are consistent with recent progress on experimental research into UBPs along with our previous calculations on the d5SICS-dNaM pair and, therefore, strengthen the hypothesis that hydrogen bonding might not be absolutely essential and that interbase stacking dispersion interactions play a key role in the stabilization of genetic materials.

Iluminated by foreign letters - Strategies for site-specific cyclopropene modification of large functional RNAs via in vitro transcription.

The synthesis of sequence-specifically modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods is experimentally challenging. We are using a new approach based on an expanded genetic alphabet preparing site-specifically modified RNA molecules via standard in vitro transcription. In this report, the site-specific labeling of functional RNAs, in particular ribozymes and a long non-coding RNA with cyclopropene moieties, is presented. We provide detailed instructions for RNA labeling via in vitro transcription and include required analytical methods to verify production and identity of the transcript. We further present post-transcriptional inverse electron demand Diels-Alder cycloaddition reactions on the cyclopropene-modified sequences and discuss applications of the genetic alphabet expansion transcription for in vitro preparation of labeled functional RNAs with complex foldings. In detail, the glmS and CPEB3 ribozymes were site-specifically decorated with methyl cyclopropene moieties using the unnatural TPT3CP triphosphate and were proven to be still functional. In addition, the structurally complex A region of the Xist lncRNA (401nt) was site-specifically modified with methyl cyclopropene and detected by fluorescence after cycloaddition reaction with a tetrazine-BODIPY conjugate.

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required.

Enhanced Stability of DNA Nanostructures by Incorporation of Unnatural Base Pairs.

Self-assembled DNA nanostructures hold great promise in the fields of nanofabrication, biosensing and nanomedicine. However, the inherent low stability of the DNA double helices, formed by weak interactions, largely hinders the assembly and functions of DNA nanostructures. In this study, we redesigned and constructed a six-arm DNA junction by incorporation of the unnatural base pairs 5-Me-isoC/isoG and A/2-thioT into the double helices. They not only retained the structural integrity of the DNA nanostructure, but also showed enhanced thermal stability and resistance to T7 Exonuclease digestion. This research may expand the applications of DNA nanostructures in nanofabrication and biomedical fields, and furthermore, the genetic alphabet expansion with unnatural base pairs may enable us to construct more complicated and diversified self-assembled DNA nanostructures.

A DNA Structure Containing AgI -Mediated G:G and C:C Base Pairs.

Metal-mediated base pairs have been extensively utilized in many research fields, including genetic-code extension, novel therapeutics development, and nanodevice design. Compared to other cations, AgI is more flexible in pairing with natural base pairs. Herein, we present a DNA structure containing two C-AgI -C pairs and the first reported G-AgI -G pair in a short 8mer DNA strand. This structure not only provides detailed insight into these AgI -mediated base-pairing patterns in DNA, but also represents the first nonhelical DNA structure driven by heavy-metal ions, thus further contributing to the structural diversity of DNA. This unique complex structure is highly sequence-dependent, thus implying functional potentials as a new DNA aptamer that can bind and recognize silver ions. These results not only advance our understanding of the interactions between AgI and nucleobases, but also provide a unique structural component for the rational design of new DNA nanodevices.

Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?

In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant reduction in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near-visible radiation. The photosensitizing properties of the lowest-energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited-state mechanisms by which near-visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady-state and time-resolved spectroscopies are combined with quantum-chemical calculations in order to reveal the excited-state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in solution. It is shown that excitation of d5SICS or dNaM with near-visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomolecules within the cellular environment.

Faithful PCR Amplification of an Unnatural Base-Pair Analogue with Four Hydrogen Bonds.

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN(N):NaO(O)), having four hydrogen bonds was investigated. Kinetic studies of single-nucleotide insertion revealed that ImN(N) and NaO(O) were recognized as complementary bases by an exonuclease-deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN(O):NaO(N) and ImO(N):NaN(O)) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen-bonding pattern of the ImN(N):NaO(O) pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN(N):NaO(O) pair was achieved by using DNA polymerases possessing 3'→5' exonuclease activity (≈99.5 % per doubling).

Replicating an expanded genetic alphabet in cells.

Recent advances in synthetic biology have made it possible to replicate an unnatural base pair in living cells. This study highlights the technologies developed to create a semisynthetic organism with an expanded genetic alphabet and the potential challenges of moving forward.