Aptamer Screening: Current Methods and Future Trend towards Non-SELEX Approach.

Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy.

A novel XNA-based Luminex assay to detect UBA1 somatic mutations associated with VEXAS syndrome.

Objectives: Patients with VEXAS syndrome carry mutations of UBA1 gene coding for the E1 enzyme. The three most frequent mutations are p.M41T(122T > C), p.M41V (c.121A > G), and p.M41L (c.121A > C) in codon 41 of exon 3. Currently, sanger sequencing was mainly used to detect these mutations, which has low sensitivity and low throughput. There is a need of high sensitivity, simple and high throughput method to characterize patients with VEXAS syndrome. Methods: Based on our proprietary XNA technology, we have developed a QClamp® Plex platform to detect eight mutations in a single reaction using the Luminex xMap technology. The assay sensitivity, specificity and precision were subsequently evaluated. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive DNA samples and the sanger sequencing method was used for comparison. Results: With spiking synthetic mutant DNA in wildtype GM24385 cell line DNA, this assay can detect UBA1 mutations with a detection limit of variant allele frequency (VAF) at 0.1-5%. Our assay shows 100% concordance with Sanger sequencing results when used for analyzing 15 positive and 19 negative clinical samples. Conclusions: The QClamps® Plex UBA1 Mutation Detection Assay is a quicker, simpler, and more sensitive assay that can accurately detect the UBA1 mutations even at early stages with low mutation frequency.

Impact of Single Acyclic Phosphonate Nucleotide (ZNA) Modifications on DNA Duplex Stability.

An acyclic phosphonate-linked nucleic acid backbone (ZNA) demonstrated the capability to support duplex formation and propagate genetic information in vivo, unveiling its potential for evolution into a synthetic genetic system (XNA). To determine the structural impact of such modification, modified Dickerson Drew DNA dodecamers (DDDs) were prepared by solid phase synthesis, each containing either an (R) or (S) isomeric form of a cytosine ZNA nucleotide. While the DDD is known to adopt a stable duplex, both duplex and hairpin forms were simultaneously observed for both modified oligonucleotides by NMR spectroscopy over a broad temperature range (5-65 °C). Diffusion-ordered spectroscopy (DOSY) experiments allowed to separate duplex and hairpin signals based on the different diffusion constants of both conformational states. For the oligomer containing (R)-ZNA, only the duplex form occurred at 5 °C, while it was not possible to determine by NMR a single hairpin conformation at higher temperatures. In the case of the (S)-ZNA nucleoside modified oligomer, both hairpin and duplex forms were observable at 0 °C, while a single hairpin conformation was detected at 37 °C, suggesting a higher destabilizing effect on dsDNA.

A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer.

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5-7%, 1-2%, and 1-2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

The Use of Xenonucleic Acids Significantly Reduces the In Vivo Drift of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant "xenonucleic acid" 2'O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2'O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2'O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2'O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.

Engineering TNA polymerases through iterative cycles of directed evolution.

DNA polymerases are important tools for biotechnology, synthetic biology, and chemical biology as they are routinely used to amplify and edit genetic information. However, natural polymerases do not recognize artificial genetic polymers (also known as xeno-nucleic acids or XNAs) with unique sugar-phosphate backbone structures. Directed evolution offers a possible solution to this problem by facilitating the discovery of engineered versions of natural polymerases that can copy genetic information back and forth between DNA and XNA. Here we report a directed evolution strategy for discovering polymerases that can synthesize threose nucleic acid (TNA) on DNA templates. The workflow involves library generation and expression in E. coli, high-throughput microfluidics-based screening of uniform water-in-oil droplets, plasmid recovery, secondary screening, and library regeneration. This technique is sufficiently general that it could be applied to a wide range of problems involving DNA modifying enzymes.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

The Expanded Central Dogma: Genome Resynthesis, Orthogonal Biosystems, Synthetic Genetics.

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

Crystal structures of 'ALternative Isoinformational ENgineered' DNA in B-form.

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host-guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Selection of RNA-Cleaving TNA Enzymes for Cellular Mg2+ Imaging.

Catalytic DNA-based fluorescent sensors have enabled cellular imaging of metal ions such as Mg2+ . However, natural DNA is prone to nuclease-mediated degradation. Here, we report the in vitro selection of threose nucleic acid enzymes (TNAzymes) with RNA endonuclease activities. One such TNAzyme, T17-22, catalyzes a site-specific RNA cleavage reaction with a kcat of 0.017 min-1 and KM of 675 nM. A fluorescent sensor based on T17-22 responds to an increasing concentration of Mg2+ with a limit of detection at 0.35 mM. This TNAzyme-based sensor also allows cellular imaging of Mg2+ . This work presents the first proof-of-concept demonstration of using a TNA catalyst in cellular metal ion imaging.

XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification.

N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modifications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypomethylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis.

Tailored Tolane-Perfluorotolane Assembly as Supramolecular Base Pair Replacement in DNA.

Arene-fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane-perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π-stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene-fluoroarene-programmed assembly of DNA.

Reverse Transcriptases: From Discovery and Applications to Xenobiology.

Reverse transcriptases are DNA polymerases that can use RNA as a template for DNA synthesis. They thus catalyze the reverse of transcription. Although discovered in 1970, reverse transcriptases are still of great interest and are constantly being further developed for numerous modern research approaches. They are frequently used in biotechnological and molecular diagnostic applications. In this review, we describe the discovery of these fascinating enzymes and summarize research results and applications ranging from molecular cloning, direct virus detection, and modern sequencing methods to xenobiology.

Chemical Modifications for a Next Generation of Nucleic Acid Aptamers.

In the past three decades, in vitro systematic evolution of ligands by exponential enrichment (SELEX) has yielded many aptamers for translational applications in both research and clinical settings. Despite their promise as an alternative to antibodies, the low success rate of SELEX (∼30 %) has been a major bottleneck that hampers the further development of aptamers. One hurdle is the lack of chemical diversity in nucleic acids. To address this, the aptamer chemical repertoire has been extended by introducing exotic chemical groups, which provide novel binding functionalities. This review will focus on how modified aptamers can be selected and evolved, with illustration of some successful examples. In particular, unique chemistries are exemplified. Various strategies of incorporating modified building blocks into the standard SELEX protocol are highlighted, with a comparison of the differences between pre-SELEX and post-SELEX modifications. Nucleic acid aptamers with extended functionality evolved from non-natural chemistries will open up new vistas for function and application of nucleic acids.

In Vivo Assembly and Expression of DNA Containing Non-canonical Bases in the Yeast Saccharomyces cerevisiae.

Chemically modified nucleic acids are of utmost interest in synthetic biology for creating a regulable and sophisticated synthetic system with tailor-made properties. Implanting chemically modified nucleic acids in microorganisms might serve biotechnological applications, while using them in human cells might lead to new advanced medicines. Previously, we reported that a fully modified DNA sequence (called DZA) composed of the four base-modified nucleotides - 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine and 5-fluorocytosine - could function as a genetic template in prokaryotic cells, Escherichia coli. Here, we report the synthesis of long, partially, or fully modified DZA fragments that encode the yeast-enhanced red fluorescent protein (yEmRFP). The DZA sequences were directly introduced in the genome of the eukaryotic cells, Saccharomyces cerevisiae, via the yeast natural homologous recombination machinery. The simple and straightforward DZA cloning strategy reported here might be of interest to scientists working in the field of xenobiology in yeast.

Xeno-Nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding.

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers.

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.

Stable Hairpin Structures Formed by Xylose-Based Nucleic Acids.

Xenobiology explores synthetic nucleic acid polymers as alternative carriers of genetic information to expand the central dogma. The xylo- and deoxyxylo-nucleic acids (XyNA and dXyNA), containing 3' epimers of riboses and deoxyriboses, are considered to be potential candidates for an orthogonal system. In this study, thermal and spectroscopic analyses show that XyNA and dXyNA form stable hairpins. The dXyNA hairpin structure determined by NMR spectroscopy contains a flexible loop that locks the stem into a stable ladder-like duplex with marginal right-handed helicity. The reduced flexibility of the dXyNA duplex observed in the stem of the hairpin demonstrates that folding of dXyNA yields more stable structures described so far.

Dual Crosslinking Photo-Switches for Orthogonal Photo-Control of Hybridization Between Serinol Nucleic Acid and RNA.

Wavelength-selective photo-regulation by multiple chromophores responding to different wavelengths can expand the variation of photo-manipulating systems. Herein, we report the orthogonal photo-regulation of duplex formation between serinol nucleic acid (SNA) and RNA using light-induced crosslinking reactions mediated by a new photo-reactive nucleobase 8-naphthylvinyladenine (NV A) and previously described 8-pyrenylvinyladenine (PV A). An intrastrand crosslink was induced in an SNA strand containing two adjacent NV A residues by irradiation with 340-405 nm light; the crosslink was reversed by irradiation with ≤300 nm light. In an SNA strand with adjacent NV A and PV A residues, an intrastrand crosslink resulted from irradiation with 405-465 nm light that was reversed by irradiation with ≤340 nm light. Intrastrand photo-crosslinking caused severe destabilization of an SNA/RNA duplex, resulting in dissociation to single strands. Cycloreversion resulted in duplex formation. With these NV A/NV A and NV A/PV A photo-switches, four hybridization states of two SNA/RNA duplexes could be orthogonally photo-controlled by irradiation with a suitable wavelength of light.