Unnatural Base Pairs Research Articles

Six-Letter DNA Nanotechnology: Incorporation of Z-P Base Pairs into Self-Assembling 3D Crystals.

Artificially expanded genetic information systems (AEGIS) were developed to expand the diversity and functionality of biological systems. Recent experiments have shown that these expanded DNA molecular systems are robust platforms for information storage and retrieval as well as useful for basic biotechnologies. In tandem, nucleic acid nanotechnology has seen the use of information-based "semantomorphic" encoding to drive the self-assembly of a vast array of supramolecular devices. To establish the effectiveness of AEGIS toward nanotechnological applications, we investigated the ability of a six-letter alphabet composed of A:T, G:C and synthetic Z:P (Z, 6-amino-3-(1'-β-d-2'-deoxy ribofuranosyl)-5-nitro-(1H)-pyridin-2-one; P, 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo-[1,2a]-1,3,5-triazin-(8H)-4-one) base pairs to engage in 3D self-assembly. We found that crystals could be programmably assembled from AEGIS oligomers. We conclude that unnatural base pairs can be used for the topological self-assembly of crystals. We anticipate the expansion of AEGIS-based nucleic acid nanotechnologies to enable the development of novel nanomaterials, high-fidelity signal cascades, and dynamic nanoscale devices.

Investigation of the Nonradiative Photoprocesses of Unnatural DNA Base: 7-(2-Thienyl)-imidazo[4,5-b]pyridine (Ds)─A Computational Study.

7-(2-Thienyl)-imidazo[4,5-b]pyridine (Ds) is an unnatural nucleic acid that forms a stable pair with pyrrole-2-carbaldehyde (Pa) in DNA. This Ds-Pa pair gets stabilized via van der Waals interaction and shape fitting. In our previous study [Ghosh, P. J. Phys. Chem. A 2021, 125, 5556-5561], we investigated the nonradiative photoprocesses of the unnatural DNA base Pa, and also there are some studies on its stability and reactivity in the ground state. But, to consider it as a good unnatural base pair, one has to understand its stability not only in the ground state but also in the excited states after absorbing ultraviolet (UV) radiation. Therefore, in this study, the excited-state photoprocesses of Ds on UV irradiation and its nonradiative decay channels have been investigated using state-of-the-art multireference methods, and this investigation finally leads the molecule to access the minimum energy crossing point (MECP) via a downhill pathway.

Improved synthesis of the unnatural base NaM, and evaluation of its orthogonality in in vitro transcription and translation.

Unnatural base pairs (UBP) promise to diversify cellular function through expansion of the genetic code. Some of the most successful UBPs are the hydrophobic base pairs 5SICS:NaM and TPT3:NaM developed by Romesberg. Much of the research on these UBPs has emphasized strategies to enable their efficient replication, transcription and translation in living organisms. These experiments have achieved spectacular success in certain cases; however, the complexity of working in vivo places strong constraints on the types of experiments that can be done to optimize and improve the system. Testing UBPs in vitro, on the other hand, offers advantages including minimization of scale, the ability to precisely control the concentration of reagents, and simpler purification of products. Here we investigate the orthogonality of NaM-containing base pairs in transcription and translation, looking at background readthrough of NaM codons by the native machinery. We also describe an improved synthesis of NaM triphosphate (NaM-TP) and a new assay for testing the purity of UBP containing RNAs.

Recognition and Sequencing of Mutagenic DNA Adduct at Single-Base Resolution Through Unnatural Base Pair.

DNA lesions are linked to cancer, aging, and various diseases. The recognition and sequencing of special DNA lesions are of great interest but highly challenging. In this paper, an unnatural-base-pair-promoting method for sequencing highly mutagenic ethenodeoxycytidine (εC) DNA lesions that occurred frequently is developed. First, a promising unnatural base pair of dεC-dNaM to recognize εC lesions is identified, and then a conversion PCR is developed to site-precise transfer dεC-dNaM to dTPT3-dNaM for convenient Sanger sequencing. The low sequence dependence of this method and its capacity for the enrichment of dεC in the abundance of as low as 1.6 × 10-6 nucleotides is also validated. Importantly, the current method can be smoothly applied for recognition, amplification, enrichment, and sequencing of the real biological samples in which εC lesions are generated in vitro or in vivo, thus offering the first sequencing methodology of εC lesions at single-base resolution. Owing to its simple operations and no destruction of inherent structures of DNA, the unnatural-base-pair strategy may provide a new platform to produce general tools for the sequencing of DNA lesions that are hardly sequenced by traditional strategies.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

Expansion of Genetic Alphabets: Designer Nucleobases and Their Applications

AbstractAll living things use DNA and RNA to store, retrieve, and transmit their genetic information. The complementary Watson–Crick nucleobase-pairs (A/T and G/C base-pairs), have been documented for years as being essential for the integrity of the DNA double helix and also for replication and transcription. With only four poorly fluorescent naturally occurring nucleic acid bases (namely A, G, T/U, and C), the extraction of genetic information is difficult. Further, the chemical diversity of DNA and RNA is severely limited. Deoxyribose/ribose-phosphate backbones also constrain DNA and RNA characteristics and have poor chemical and physiological stability, which significantly restricts the practical applications of DNA and RNA. Over the years, extensively modified nucleobase pairs with novel base-pairing properties have been synthesized. Such designer nucleobases, serving as an expanded genetic alphabet, have been used for the design and synthesis of DNA and RNA analogues with tailored informational/functional properties. Recent developments in the production of synthetic unnatural base pairs pave the way for xenobiology research and genetic alphabet expansion technology. In this review, we present a brief history of the development of several hydrogen- and non-hydrogen-bonded unnatural base pairs and their applications. We also highlight our work in designing and synthesizing a new class of triazolyl unnatural nucleosides that offer a unique charge-transfer (CT) complexation force towards stabilizing DNA-duplexes when incorporated into short oligonucleotide sequences.

Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

Reverse transcription as key step in RNA in vitro evolution with unnatural base pairs

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. Reverse transcription of UBPs as key step during RNA in vitro selection is investigated.

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach.

Incorporation of artificial 3rd base pairs (unnatural base pairs, UBPs) has emerged as a fundamental technique in pursuit of expanding the genetic alphabet. 2,6-Dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN), a potential unnatural base pair (UBP) developed by Romesberg and colleagues, has been shown to have remarkable capability for replication within DNA. Crystal structures of a Taq polymerase/double-stranded DNA (ds-DNA) complex containing a DS-DN pair in the 3' terminus showed a parallelly stacked geometry for the pre-insertion, and an intercalated geometry for the post-insertion structure. Unconventional orientations of DS-DN inside a DNA duplex have inspired scientists to investigate the conformational orientations and structural properties of UBP-incorporated DNA. In recent years, computational simulations have been used to investigate the geometry of DS-DN within the DNA duplex; nevertheless, unresolved questions persist owing to inconclusive findings. In this work, we investigate the structural and dynamical properties of DS and DN inside a ds-DNA strand in aqueous solution considering both short and long DNA templates using polarizable, and non-polarizable classical MD simulations. Flexible conformational change of UBP with major populations of Watson-Crick-Franklin (WCF) and three distinct non-Watson-Crick-Franklin (nWCFP1, nWCFP2, nWCFO) conformations through intra and inter-strand flipping have been observed. Our results suggest that a dynamical conformational change leads to the production of diffierent conformational distribution for the systems. Simulations with a short ds-DNA duplex suggest nWCF (P1 and O) as the predominant structures, whereas long ds-DNA duplex simulations indicate almost equal populations of WCF, nWCFP1, nWCFO. DS-DN in the terminal position is found to be more flexible with occasional mispairing and fraying. Overall, these results suggest flexibility and dynamical conformational change of the UBP as well as indicate varied conformational distribution irrespective of starting orientation of the UBP and length og DNA strand.

Orchestration and theranostic applications of synthetic genome with Hachimoji bases/building blocks.

Synthetic genomics is a novel field of chemical biology where the chemically modified genetic alphabets have been considered in central dogma of life. Tweaking of chemical compositions of natural nucleotide bases could be developed as novel building blocks of DNA/RNA. The modified bases (dP, dZ, dS, and dB etc.) have been demonstrated to be adaptable for replication, transcription and follow Darwinism law of evolution. With advancement of chemical biology especially nucleotide chemistry, synthetic genetic codes have been discovered and Hachimoji nucleotides are the most important and significant one among them. These additional nucleotide bases can form orthogonal base-pairing, and also follow Darwinian evolution and other structural features. In the Hachimoji base pairing, synthetic building blocks are formed using eight modified nucleotide (DNA/RNA) letters (hence the name "Hachimoji"). Their structural conformations, like polyelectrolyte backbones and stereo-regular building blocks favor thermodynamic stability and confirm Schrodinger aperiodic crystal. From the structural genomics aspect, these synthetic bases could be incorporated into the central dogma of life. Researchers have shown Hachimoji building blocks were transcribed to its RNA counterpart as a functional fluorescent Hachimoji aptamer. Apart from several unnatural nucleotide base pairs maneuvered into its invitro and invivo applications, this review describes future perspective towards the development and therapeutic utilization of the genetic codes, a primary objective of synthetic and chemical biology.

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

Systematic Mutation and Unnatural Base Pair Incorporation Improves Riboswitch-Based Biosensor Response Time.

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance of other RNA-based tools.

One-Pot Enzymatic Preparation of Oligonucleotides with an Expanded Genetic Alphabet via Controlled Pause and Restart of Primer Extension: Making Unnatural Out of Natural.

The genetic alphabet of life has been dramatically expanded via the development of unnatural base pairs (UBPs) that work as efficiently as natural base pairs in the storage and retrieval of genetic information. Among the most predominant UBPs, dNaM-dTPT3 and its analogues have been successfully employed to build semisynthetic cells with a functional six-letter genome. With the rapidly growing applications of UBPs in vitro and in vivo, there is an ever-increasing demand for DNA oligonucleotides containing unnatural bases (UBs) at desired positions. Conventional solid-phase synthesis of oligonucleotides has intrinsic limitations and needs to use unstable unnatural phosphoramidites and a DNA synthesizer, so it does not meet the daily urgent requirement for a few UB-containing DNA oligonucleotides in the laboratory. In this work, we develop a one-pot enzymatic method for preparing dNaM- or dTPT3-containing DNA oligonucleotides via controlled pause and restart of primer extension mediated by Klenow fragment (exo-). By systematic optimization of the reaction conditions, high efficiencies and product purities have been achieved. The universality of this method for preparing DNA oligonucleotides containing dNaM or dTPT3 in different sequence contexts is also demonstrated. This method allows convenient production of an arbitrary UB-containing DNA oligonucleotide in a single test tube with only two natural DNA oligonucleotides, stable nucleoside triphosphates, Klenow fragment (exo-), and other common reagents in the laboratory, providing the lowest cost and the highest simplicity for the enzymatic preparation of UB-containing oligonucleotides. Clearly, this method has great potential to facilitate the in vitro and in vivo applications of the UBPs.

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.

Recognition of an Unnatural Base Pair by Tool Enzymes from Bacteriophages and Its Application in the Enzymatic Preparation of DNA with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet in vitro and in vivo. UBP dNaM-dTPT3 and its analogues have been successfully used to construct the first set of semi-synthetic organisms, which suggested the great potential of UBPs to be used for producing novel synthetic biological parts. Two prerequisites for doing so are the facile manipulation of DNA containing UBPs with common tool enzymes, including DNA polymerases and ligases, and the easy availability of UBP-containing DNA strands. Besides, for the application of UBPs in phage synthetic biology, the recognition of UBPs by phage enzymes is essential. Here, we first explore the recognition of dNaM-dTPT3 by a family B DNA polymerase from bacteriophage, T4 DNA polymerase D219A. Results from primer extension, steady-state kinetics, and gap-filling experiments suggest that T4 DNA polymerase D219A can efficiently and faithfully replicate dNaM-dTPT3, and efficiently fill a gap by inserting dTPT3TP or its analogues opposite dNaM. We then systematically explore the recognition of dNaM-dTPT3 and its analogues by different DNA ligases from bacteriophages and find that these DNA ligases are generally able to efficiently ligate the DNA nick next to dNaM-dTPT3 or its analogues, albeit with slightly different efficiencies. These results suggest more enzymatic tools for the manipulation of dNaM-dTPT3 and indicate the potential use of dNaM-dTPT3 for expanding the genetic alphabet in bacteriophages. Based on these results, we next develop and comprehensively optimize an upgraded method for enzymatic preparation of unnatural nucleobase (UB)-containing DNA oligonucleotides with good simplicity and universality.

Functional nucleic acids with synthetic sugar or nucleobase moieties

Functional nucleic acids including aptamers and DNAzymes are a class of valuable molecular tool for biotechnology. However, DNA and RNA aptamers and catalysts suffer from low biological stability and limited chemical diversity. Xeno-nucleic acids (XNAs) refer to nucleic acid analogues containing sugar moieties that are structurally distinct from DNA and RNA and possess advantageous properties. In this article, we first focus on two types of XNAs, threose nucleic acid (TNA) and 2’-fluoroarabinose nucleic acid (FANA), and summarize recent in vitro selections of TNA and FANA aptamers and catalysts. We then review three classes of unnatural base pairs (UBPs) and highlight examples of UBP-containing DNA aptamers and DNAzymes. Lastly, we briefly describe an XNA-modified DNAzyme 10–23 (X10-23) and its application in RNA knockdown and virus detection. Functional XNAs provide important chemical biology tools for biomedical research and future interdisciplinary collaboration will boost XNA basic research and clinical translation.

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.

Constructing Artificial Nucleobase Compilation to Enable Precise Molecular Medicine†

Comprehensive SummaryNucleic acids are the hereditary information storage medium of life. Due to its high programmability and good biocompatibility, the applications of nucleic acids are not limited to their natural function. However, the efficiency of nucleic acids is often hampered by inherent limitations in numerous practical applications, including limited access to complex functional patterns due to only four building blocks, facile degradation by nucleases, rapid renal clearance, poor pharmacokinetic properties, and so on. To end this, the researchers developed unnatural base pairs (UBPs) and numerous artificial analogues of nucleosides and oligonucleotides in recent decades. The developed UBPs and nucleoside base analogues together constitute artificial nucleobase compilation, which promotes the development of biomedical sciences. Here, we describe the development of artificial nucleobase compilation and summarized its applications in precise molecular medicine, including PCR‐based diagnostics, aptamer‐based diagnostics, nucleobase analogue drugs construction, ASO modification, aptamer‐based therapeutics and biomaterials construction. This review provides an overview of current opportunities and challenges of artificial nucleobase‐related precise molecular medicine.

Locating, tracingand sequencing multiple expanded genetic letters in complex DNA context via a bridge-base approach.

A panel of unnatural base pairs is developed to expand genetic alphabets. One or more unnatural base pairs (UBPs) can be inserted to enlarge the capacity, diversity, and functionality of canonical DNA, so monitoring the multiple-UBPs-containing DNA by simple and convenient approaches is essential. Herein, we report a bridge-base approach to repurpose the capability of determining TPT3-NaM UBPs. The success of this approach depends on the design of isoTAT that can simultaneously pair with NaM and G as a bridge base, as well as the discovering of the transformation of NaM to A in absence of its complementary base. TPT3-NaM can be transferred to C-G or A-T by simple PCR assays with high read-through ratios and low sequence-dependent properties, permitting for the first time to dually locate the multiple sites of TPT3-NaM pairs. Then we show the unprecedented capacity of this approach to trace accurate changes and retention ratios of multiple TPT3-NaM UPBs during in vivo replications. In addition, the method can also be applied to identify multiple-site DNA lesions, transferring TPT3-NaM makers to different natural bases. Taken together, our work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs.