Non-natural Nucleic Acids Research Articles

A general temperature-guided language model to design proteins of enhanced stability and activity.

Designing protein mutants with both high stability and activity is a critical yet challenging task in protein engineering. Here, we introduce PRIME, a deep learning model, which can suggest protein mutants with improved stability and activity without any prior experimental mutagenesis data for the specified protein. Leveraging temperature-aware language modeling, PRIME demonstrated superior predictive ability compared to current state-of-the-art models on the public mutagenesis dataset across 283 protein assays. Furthermore, we validated PRIME's predictions on five proteins, examining the impact of the top 30 to 45 single-site mutations on various protein properties, including thermal stability, antigen-antibody binding affinity, and the ability to polymerize nonnatural nucleic acid or resilience to extreme alkaline conditions. More than 30% of PRIME-recommended mutants exhibited superior performance compared to their premutation counterparts across all proteins and desired properties. We developed an efficient and effective method based on PRIME to rapidly obtain multisite mutants with enhanced activity and stability. Hence, PRIME demonstrates broad applicability in protein engineering.

Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.

2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology.

Developing predictive hybridization models for phosphorothioate oligonucleotides using high-resolution melting.

The ability to predict nucleic acid hybridization energies has been greatly enabling for many applications, but predictive models require painstaking experimentation, which may limit expansion to non-natural nucleic acid analogues and chemistries. We have assessed the utility of dye-based, high-resolution melting (HRM) as an alternative to UV-Vis determinations of hyperchromicity in order to more quickly acquire parameters for duplex stability prediction. The HRM-derived model for phosphodiester (PO) DNA can make comparable predictions to previously established models. Using HRM, it proved possible to develop predictive models for DNA duplexes containing phosphorothioate (PS) linkages, and we found that hybridization stability could be predicted as a function of sequence and backbone composition for a variety of duplexes, including PS:PS, PS:PO, and partially modified backbones. Individual phosphorothioate modifications destabilize helices by around 0.12 kcal/mol on average. Finally, we applied these models to the design of a catalytic hairpin assembly circuit, an enzyme-free amplification method used for nucleic acid-based molecular detection. Changes in PS circuit behavior were consistent with model predictions, further supporting the addition of HRM modeling and parameters for PS oligonucleotides to the rational design of nucleic acid hybridization.

Expanding the reverse transcription toolbox.

Tailor-made reverse transcriptases are used in molecular biology and synthetic genetics. However, re-engineering these enzymes to work with non-natural nucleic acids is difficult and requires powerful directed evolution strategies. Now, an adaptable selection approach has been demonstrated for the evolution of new reverse transcriptases.

Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.

Template-Directed Nonenzymatic Primer Extension Using 2-Methylimidazole-Activated Morpholino Derivatives of Guanosine and Cytidine.

Efforts to develop self-replicating nucleic acids have led to insights into the origin of life and have also suggested potential pathways to the design of artificial life forms based on non-natural nucleic acids. The template-directed nonenzymatic polymerization of activated ribonucleotide monomers is generally slow because of the relatively weak nucleophilicity of the primer 3′-hydroxyl. To circumvent this problem, several nucleic acids based on amino-sugar nucleotides have been studied, and as expected, the more-nucleophilic amine generally results in faster primer extension. Extending this logic, we have chosen to study morpholino nucleic acid (MoNA), because the secondary amine of the morpholino-nucleotides is expected to be highly nucleophilic. We describe the synthesis of 2-methylimidazole-activated MoNA monomers from their corresponding ribonucleoside 5′-monophosphates and the synthesis of an RNA primer with a terminal MoNA nucleotide. We show that the activated G and C MoNA monomers enable rapid and efficient extension of the morpholino-terminated primer on homopolymeric and mixed-sequenced RNA templates. Our results show that MoNA is a non-natural informational polymer that is worthy of further study as a candidate self-replicating material.

Mapping the affinity landscape of Thrombin-binding aptamers on 2΄F-ANA/DNA chimeric G-Quadruplex microarrays.

In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2΄-Fluoroarabinonucleic acid (2΄F-ANA) is a prime candidate for such use in microarrays. Indeed, 2΄F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2΄F-ANA and 2΄F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2΄F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2΄F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2΄F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2΄F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

The Toolbox for Modified Aptamers.

Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.

Artificially Created Nucleic Acids and Peptides/Proteins in Chemical Biology

Nucleic acids—DNA and RNA—have been chosen by Mother Nature as the key players for orchestrating the preservation, transfer, and expression of genetic information in all the biological systems on Earth. RNA has also been enlisted to carry out other important cellular functions, such as catalysis and molecular recognition. Within the hands of scientists, the function of nucleic acids has been significantly expanded beyond what is known in nature, and as a result, we are now in the possession of a large array of synthetic, nucleic acid-based catalysts (ribozymes and DNAzymes) and receptors (DNA and RNA aptamers). DNA as a genetic material itself has also been subjected to various chemical modifications in efforts to derive significantly altered or even completely new genetic systems. These systems can be used to create novel peptides and proteins that offer enhanced activities or even completely new properties over their natural protein counterparts. Furthermore, many artificially engineered nucleic acids and proteins have found useful applications as biosensors, diagnostic agents, and therapeutic drugs. This special issue is assembled to reflect recent progress in the important research arena of artificially engineered nucleic acids and proteins. This issue comprises 10 reviews and 7 research articles that can be grouped into three sections. The first section deals mainly with research on xeno-nucleic acids (XNAs)—nonnatural nucleic acid analogs with significantly altered sugar and/or phosphate backbones. D.-A. Catana et al. provide a review on the use of dinucleotides of dioxaphosphorinane-constrained nucleic acids (CNAs) to tune nucleic acid structures. This is followed by a review by E. Rozners on recent advances in chemical modifications of peptide nucleic acids (PNAs). G. Upert et al. then present a research article on designing cyclic and hairpin PNAs as inhibitors for HIV replication. Z. Wang et al. also present a research article where PNA probes were utilized for live cell imaging of mRNA expression. In their research article, T. Yamamoto et al. examine the gene-silencing effect of bridged/locked nucleic acids (BNA/LNAs). This section is closed out with a research article by S. Saxena et al. in which the molecular crowding effect on the structure and function of RecG (a helicase) was examined. The second section includes four reviews and three research articles discussing the creation of novel peptides, proteins, transfer RNAs, and peptide mimicries using various selection or screening methods. K. Fukunaga and M. Taki provide a review on the phage display technique with a particular focus on tips for conducting successful phage display experiments. Within the same topic, T. Matsubara reviews the use of phage display for the creation of carbohydrate-mimetic peptides. These are followed by a research article by T. Sumida et al. exploiting the mRNA display technique for the selection of anti-p53 Fab fragments. There are two papers concerning the in vitro compartmentalization (IVC) technique, which offers an excellent way to link a genotype to a phenotype in a physically confined environment: the first is a review article by T. Nishikawa et al. on evolving proteins using IVC and the second is a research article by A. Ogawa et al. where IVC was used to select functional transfer RNAs. T. Kawakami and H. Murakami discuss, in their review article, potential applications of a translation system with a reprogrammed genetic code to prepare a peptide mimetic library. Finally, J. K. Pokorski and D. H. Appella present an on-bead screening approach to create peptide mimicries. The last section of the issue consists of four review papers on the selection and application of functional nucleic acids. M. McKeague and M. C. DeRosa survey DNA and RNA aptamers derived by SELEX (systematic evolution of ligands via exponential enrichment) for small molecule binding, along with the compilation of nearly 40 improved methodologies of SELEX. K. Tram et al. review the application of fluorescently dressed RNA-cleaving DNAzymes for biosensing. Y. M. Chang et al. discuss the cell-SELEX technique for biomarker discovery. In addition, Y. Kasahara and M. Kuwahara provide a review on SELEX experiments exploring chemically modified nucleic acid libraries. The ultimate goal in this research arena is to generate scientific knowledge and produce new technologies that will enable the engineering of novel or improved molecular systems to control biological activities at will. Along the way, researchers may help shine light on one of the biggest mysteries surrounding the origin and evolution of life on Earth—the question of why Mother Nature has selected 4 nucleotides to construct nucleic acids and 20 amino acids to construct proteins. We hope this special issue can further spur research efforts in this area.

Artificial DNA and surface plasmon resonance

The combined use of surface plasmon resonance (SPR) and modified or mimic oligonucleotides have expanded diagnostic capabilities of SPR-based biosensors and have allowed detailed studies of molecular recognition processes. This review summarizes the most significant advances made in this area over the past 15 years. Functional and conformationally restricted DNA analogs (e.g., aptamers and PNAs) when used as components of SPR biosensors contribute to enhance the biosensor sensitivity and selectivity. At the same time, the SPR technology brings advantages that allows forbetter exploration of underlying properties of non-natural nucleic acid structures such us DNAzymes, LNA and HNA.

Non-natural nucleic acids for synthetic biology

Genetic manipulation is an important facet of synthetic biology but can be complicated by undesired nuclease degradation. Incorporating non-natural nucleic acids into a gene could convey resistance to nucleases and promote expression. The compatibility of non-natural nucleosides with polymerases is reviewed with a focus on results from the past two years. Details are provided about how the different systems could be useful in synthetic biology.

Inhibition of influenza virus infection by targeting genome conserved region with non-natural nucleic acid

Two highly conserved 15 base sequences of influenza A virus genome were identified by CONSERV software, which can detect contiguous conserved sequences of biological sequences. Antiviral effect of phosphorothioate oligonucleotide that target these conserved sequences was evaluated by plaque formation assay and cell viability assay. Pre-treatment of cells with anti-PB2 (RNA polymerase subunit) reduced the size of plaques in a sequence dependent manner. Post-treatment of cells with anti-PB2 phosphorothioate oligonucleotide inhibited the virus-induced cytotoxicity.

PNA−DNA Duplexes, Triplexes, and Quadruplexes Are Stabilized with trans-Cyclopentane Units

Peptide nucleic acids (PNAs) are non-natural nucleic acid mimics that bind to complementary DNA and RNA with high affinity and selectivity. PNA can bind to nucleic acids in a number of different ways. Currently, the formation of PNA-oligonucleotide duplex, triplex, and quadruplex structures have been reported. PNAs have been used in numerous biomedicial applications, but there are few strategies to predictably improve the binding properties of PNAs by backbone modification. We have been studying the benefits of incorporating (S,S)-trans-cyclopentane diamine units (tcyp) into the PNA backbone. In this Communication, we report the improvement in stability associated with tcyp incorporation into PNA-DNA duplexes, triplexes, and quadruplexes. The broad utility of this modification across multiple types of PNA structures is unique and should prove useful in the development of applications that rely on PNA.

Modified nucleic acids on display

Non-natural nucleic acids have offered insight into natural DNA and RNA and provided experimental systems to test theories of nucleic acid evolution. Numerous structurally modified nucleic acids have been prepared in the laboratory that show a diverse range of genetic and structural properties. One in particular, threose nucleic acid (TNA), has attracted attention because of its chemical simplicity and its ability to base-pair with complementary DNA, RNA and other TNA. Ichida et al. now show, in the Journal of the American Chemical Society, that long stretches of TNA can be synthesized by an engineered DNA polymerase, and they suggest a system for the in vitro selection of TNA molecules with novel functions.

Two dimensional PNA/DNA arrays: estimating the helicity of unusual nucleic acid polymers.

We extend the generality of nucleic acid-based structural nanotechnology by incorporating non-natural nucleic acids into a DNA double crossover (DX) molecule; visualizing two-dimensional arrays of these DX molecules by Atomic Force Microscopy (AFM) enables us to measure the helical repeat of any heteroduplex sequence capable of forming the outer arms of a DX.