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.

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.

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.

Synthesis, Reverse Transcription, Replication, and Inter-Transcription of 2'-Modified Nucleic Acids with Evolved Thermophilic Polymerases: Efforts toward Multidimensional Expansion of the Central Dogma.

In the past decades, various xenobiotic nucleic acids (XNAs), including 2'-modified nucleic acids, have been developed as novel genetic materials and demonstrated great potential in synthetic biology and biotechnology. Enzymatic polymerization and replication of these artificial polymers are obviously the prerequisite to make full use of them, and DNA and RNA polymerases from different families have thus been extensively engineered for these purposes. However, the performance of engineered XNA polymerases is still far from satisfactory, especially in terms of the efficiency of synthesizing XNA with bigger lengths and the capability of directly replicating XNAs or transcribing one XNA to another. In this work, we tailored a mutant of Stoffel fragment of Taq DNA polymerase, SFM4-3, by engineering a key residue pair on the surfaces of fingers and thumb domains, and successfully obtained mutants with significantly enhanced efficiency for the synthesis of fully 2'-OMe-modified DNA with bigger lengths. Remarkably, we also found that these polymerase mutants are capable of synthesizing, reverse transcribing, and even replicating RNA and different fully 2'-modified XNAs, as well as transcribing one of these nucleic acids to another, with varied efficiencies. The application of these activities for producing DNA strands end-protected by XNA duplexes was then demonstrated. These results clearly suggest that the genetic information can be stored in and transmitted among DNA, RNA, and different 2'-modified XNAs with the assistance of polymerase mutants, and the central dogma of life can be expanded to higher dimensions via the development of XNAs together with engineering their polymerases.

Pan-Chromosome and Comparative Analysis of Agrobacterium fabrum Reveal Important Traits Concerning the Genetic Diversity, Evolutionary Dynamics, and Niche Adaptation of the Species.

Agrobacterium fabrum has been critical for the development of plant genetic engineering and agricultural biotechnology due to its ability to transform eukaryotic cells. However, the gene composition, evolutionary dynamics, and niche adaptation of this species is still unknown. Therefore, we established a comparative genomic analysis based on a pan-chromosome data set to evaluate the genetic diversity of A. fabrum. Here, 25 A. fabrum genomes were selected for analysis by core genome phylogeny combined with the average nucleotide identity (ANI), amino acid identity (AAI), and in silico DNA-DNA hybridization (DDH) values. An open pan-genome of A. fabrum exhibits genetic diversity with variable accessorial genes as evidenced by a consensus pan-genome of 12 representative genomes. The genomic plasticity of A. fabrum is apparent in its putative sequences for mobile genetic elements (MGEs), limited horizontal gene transfer barriers, and potentially horizontally transferred genes. The evolutionary constraints and functional enrichment in the pan-chromosome were measured by the Clusters of Orthologous Groups (COG) categories using eggNOG-mapper software, and the nonsynonymous/synonymous rate ratio (dN/dS) was determined using HYPHY software. Comparative analysis revealed significant differences in the functional enrichment and the degree of purifying selection between the core genome and non-core genome. We demonstrate that the core gene families undergo stronger purifying selection but have a significant bias to contain one or more positively selected sites. Furthermore, although they shared similar genetic diversity, we observed significant differences between chromosome 1 (Chr I) and the chromid in their functional features and evolutionary constraints. We demonstrate that putative genetic elements responsible for plant infection, ecological adaptation, and speciation represent the core genome, highlighting their importance in the adaptation of A. fabrum to plant-related niches. Our pan-chromosome analysis of A. fabrum provides comprehensive insights into the genetic properties, evolutionary patterns, and niche adaptation of the species. IMPORTANCE Agrobacterium spp. live in diverse plant-associated niches such as soil, the rhizosphere, and vegetation, which are challenged by multiple stressors such as diverse energy sources, plant defenses, and microbial competition. They have evolved the ability to utilize diverse resources, escape plant defenses, and defeat competitors. However, the underlying genetic diversity and evolutionary dynamics of Agrobacterium spp. remain unexplored. We examined the phylogeny and pan-genome of A. fabrum to define intraspecies evolutionary relationships. Our results indicate an open pan-genome and numerous MGEs and horizontally transferred genes among A. fabrum genomes, reflecting the flexibility of the chromosomes and the potential for genetic exchange. Furthermore, we observed significant differences in the functional features and evolutionary constraints between the core and accessory genomes and between Chr I and the chromid, respectively.

Thermophilic Nucleic Acid Polymerases and Their Application in Xenobiology.

Thermophilic nucleic acid polymerases, isolated from organisms that thrive in extremely hot environments, possess great DNA/RNA synthesis activities under high temperatures. These enzymes play indispensable roles in central life activities involved in DNA replication and repair, as well as RNA transcription, and have already been widely used in bioengineering, biotechnology, and biomedicine. Xeno nucleic acids (XNAs), which are analogs of DNA/RNA with unnatural moieties, have been developed as new carriers of genetic information in the past decades, which contributed to the fast development of a field called xenobiology. The broad application of these XNA molecules in the production of novel drugs, materials, and catalysts greatly relies on the capability of enzymatic synthesis, reverse transcription, and amplification of them, which have been partially achieved with natural or artificially tailored thermophilic nucleic acid polymerases. In this review, we first systematically summarize representative thermophilic and hyperthermophilic polymerases that have been extensively studied and utilized, followed by the introduction of methods and approaches in the engineering of these polymerases for the efficient synthesis, reverse transcription, and amplification of XNAs. The application of XNAs facilitated by these polymerases and their mutants is then discussed. In the end, a perspective for the future direction of further development and application of unnatural nucleic acid polymerases is provided.

Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase.

Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both in vitro and in vivo and led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, and de novo synthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma.

Nucleic acids have been extensively modified in different moieties to expand the scope of genetic materials in the past few decades. While the development of unnatural base pairs (UBPs) has expanded the genetic information capacity of nucleic acids, the production of synthetic alternatives of DNA and RNA has increased the types of genetic information carriers and introduced novel properties and functionalities into nucleic acids. Moreover, the efforts of tailoring DNA polymerases (DNAPs) and RNA polymerases (RNAPs) to be efficient unnatural nucleic acid polymerases have enabled broad application of these unnatural nucleic acids, ranging from production of stable aptamers to evolution of novel catalysts. The introduction of unnatural nucleic acids into living organisms has also started expanding the central dogma in vivo. In this article, we first summarize the development of unnatural nucleic acids with modifications or alterations in different moieties. The strategies for engineering DNAPs and RNAPs are then extensively reviewed, followed by summarization of predominant polymerase mutants with good activities for synthesizing, reverse transcribing, or even amplifying unnatural nucleic acids. Some recent application examples of unnatural nucleic acids with their polymerases are then introduced. At the end, the approaches of introducing UBPs and synthetic genetic polymers into living organisms for the creation of semi-synthetic organisms are reviewed and discussed.

Site-specific unnatural base excision via visible light.

Base excision (BE) is an important yet hard-to-control biological event. Unnatural base pairs are powerful tools to revolutionize biological studies in various areas. In this paper, we report a visible-light-induced method to construct site-specific unnatural BE and show the influence of its regulation on transcription and translation levels.

Transcription, Reverse Transcription, and Amplification of Backbone-Modified Nucleic Acids with Laboratory-Evolved Thermophilic DNA Polymerases.

Backbone-modified nucleic acids are usually more stable enzymatically than their natural counterparts, enabling their broad application as potential diagnostic or therapeutic agents. Moreover, the development of nucleic acids with unnatural backbones has expanded the pool of genetic information carriers and paved the way toward synthetic xenobiology. However, synthesizing these molecules remains very challenging due to the requirement for harsh reaction conditions and the low coupling efficiency during their traditional solid-phase synthesis. Although enzymatic synthesis provides an attractive alternative that also allows the replication and artificial evolution of these molecules, it is crucially dependent on the availability of polymerases capable of synthesizing these backbone-modified nucleotides. Previously, a series of thermostable polymerases that can efficiently synthesize or even amplify backbone-modified DNA or RNA have been evolved through a polymerase evolution method based on phage display. Herein we summarize protocols to use these evolved polymerase mutants to transcribe, reverse transcribe, and PCR amplify backbone-modified nucleic acids. We also outline the polymerase chain transcription method, developed later for the rapid production of RNA or backbone-modified RNA with one of these evolved polymerases, SFM4-3. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Transcription/synthesis of modified DNA/RNA from DNA templates with evolved polymerases SFM4-3 or SFM4-6 Basic Protocol 2: Reverse transcription of modified DNA/RNA with evolved polymerase SFM4-9 Basic Protocol 3: PCR amplification of modified DNA with evolved polymerase SFM4-3 Basic Protocol 4: Polymerase chain transcription for the production of RNA/modified RNA oligonucleotides with evolved polymerase SFM4-3.

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective.

Nucleic acids underlie the storage and retrieval of genetic information literally in all living organisms, and also provide us excellent materials for making artificial nanostructures and scaffolds for constructing multi-enzyme systems with outstanding performance in catalyzing various cascade reactions, due to their highly diverse and yet controllable structures, which are well determined by their sequences. The introduction of unnatural moieties into nucleic acids dramatically increased the diversity of sequences, structures, and properties of the nucleic acids, which undoubtedly expanded the toolbox for making nanomaterials and scaffolds of multi-enzyme systems. In this article, we first introduce the molecular structures and properties of nucleic acids and their unnatural derivatives. Then we summarized representative artificial nanomaterials made of nucleic acids, as well as their properties, functions, and application. We next review recent progress on constructing multi-enzyme systems with nucleic acid structures as scaffolds for cascade biocatalyst. Finally, we discuss the future direction of applying nucleic acid frameworks in the construction of nanomaterials and multi-enzyme molecular machines, with the potential contribution that unnatural nucleic acids may make to this field highlighted.

Selection of Aptamers with Large Hydrophobic 2'-Substituents.

Previously, we evolved a DNA polymerase, SFM4-3, for the recognition of substrates modified at their 2' positions with a fluoro, O-methyl, or azido substituent. Here we use SFM4-3 to synthesize 2'-azido-modified DNA; we then use the azido group to attach different, large hydrophobic groups via click chemistry. We show that SFM4-3 recognizes the modified templates under standard conditions, producing natural DNA and thereby allowing amplification. To demonstrate the utility of this remarkable property, we use SFM4-3 to select aptamers with large hydrophobic 2' substituents that bind human neutrophil elastase or the blood coagulation protein factor IXa. The results indicate that SFM4-3 should facilitate the discovery of aptamers that adopt novel and perhaps more protein-like folds with hydrophobic cores that in turn allow them to access novel activities.

Enzymatic Synthesis, Amplification, and Application of DNA with a Functionalized Backbone.

The ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4-3, which better tolerates 2'-modified substrates. To explore the utility of SFM4-3, we now report the characterization of its recognition of substrates with 2'-azido, 2'-chloro, 2'-amino, or arabinose sugars. We find that SFM4-3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4-3 can also PCR amplify these modified oligonucleotides. When combined with post-amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel.

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA.

There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

Selection of 2'-Fluoro-Modified Aptamers with Optimized Properties.

RNA or single-stranded DNA aptamers with 2'-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2'-modifications, we now report the selection of 2'-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity. Interestingly, the 2'-F substituents facilitate the selection of specific interactions with HNE and overcome nonspecific electrostatic interactions that can otherwise dominate. The data demonstrate that inclusion of only a few 2'-F substituents can optimize properties far beyond simple nuclease resistance and that SFM4-3 should prove valuable for the further exploration and production of aptamers with properties optimized for various applications.

Evolved polymerases facilitate selection of fully 2'-OMe-modified aptamers.

RNA or DNA aptamers with 2'-OMe-modifications have been pursued to increase resistance to nucleases, but have been difficult to identify because the OMe groups ablate polymerase recognition. We recently reported evolution of the thermostable DNA polymerases SFM4-6 and SFM4-9, which enable the efficient "transcription" and "reverse transcription", respectively, of 2'-OMe oligonucleotides. With these polymerases, we now report the first selection of fully 2'-OMe modified aptamers, specifically aptamers that bind human neutrophil elastase (HNE). Two aptamers, 2mHNE-1 and 2mHNE-2, were isolated after five rounds of selection, and four more, 2mHNE-3-6, after an additional five rounds that included selection pressure for binding in the presence of serum. All six aptamers bind with reasonable affinity, which requires the 2'-OMe substituents. Further characterization of one aptamer, 2mHNE-5, showed that unlike a previously reported natural anti-HNE aptamer, affinity for HNE is retained in the presence of high concentrations of salt or serum. The polymerases SFM4-6 and SFM4-9 should prove valuable for the production and further exploration of modified aptamers.

The expanding world of DNA and RNA.

DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe-modified oligonucleotides and their DNA counterparts via 'transcription' and 'reverse transcription' or, more importantly, that PCR-amplify partially C2'-OMe- or C2'-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.

Adaptive mutations alter antibody structure and dynamics during affinity maturation.

While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.