Genetic Alphabet Expansion Research Articles

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.

Success probability of high-affinity DNA aptamer generation by genetic alphabet expansion.

Nucleic acid aptamers as antibody alternatives bind specifically to target molecules. These aptamers are generated by isolating candidates from libraries with random sequence fragments, through an evolutionary engineering system. We recently reported a high-affinity DNA aptamer generation method that introduces unnatural bases (UBs) as a fifth letter into the library, by genetic alphabet expansion. By incorporating hydrophobic UBs, the affinities of DNA aptamers to target proteins are increased over 100-fold, as compared with those of conventional aptamers with only the natural four letters. However, there is still plenty of room for improvement of the methods for routinely generating high-affinity UB-containing DNA (UB-DNA) aptamers. The success probabilities of the high-affinity aptamer generation depend on the existence of the aptamer candidate sequences in the initial library. We estimated the success probabilities by analysing several UB-DNA aptamers that we generated, as examples. In addition, we investigated the possible improvement of conventional aptamer affinities by introducing one UB at specific positions. Our data revealed that UB-DNA aptamers adopt specific tertiary structures, in which many bases including UBs interact with target proteins for high affinity, suggesting the importance of the UB-DNA library design. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins

Amino acid sequences of proteins are encoded in nucleic acids composed of four letters, A, G, C, and T(U). However, this four-letter alphabet coding system limits further functionalities of proteins by the twenty letters of amino acids. If we expand the genetic code or develop alternative codes, we could create novel biological systems and biotechnologies by the site-specific incorporation of non-standard amino acids (or unnatural amino acids, unAAs) into proteins. To this end, new codons and their complementary anticodons are required for unAAs. In this review, we introduce the current status of methods to incorporate new amino acids into proteins by in vitro and in vivo translation systems, by focusing on the creation of new codon-anticodon interactions, including unnatural base pair systems for genetic alphabet expansion.

Dye-Conjugated Spinach RNA by Genetic Alphabet Expansion.

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

Cognate base-pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase.

We present cognate base pair selectivity in template‐dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds‐Pa. T4 DNA ligase efficiently recognizes the Ds‐Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base‐pair selectivity, relative to the hydrogen‐bond interactions between pairing bases. The efficient ligation using Ds‐Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi‐synthetic organisms containing UBPs.

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA.

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

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

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

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

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

Genetic alphabet expansion technology by creating unnatural base pairs.

Recent advancements in the creation of artificial extra base pairs (unnatural base pairs, UBPs) are opening the door to a new research area, xenobiology, and genetic alphabet expansion technologies. UBPs that function as third base pairs in replication, transcription, and/or translation enable the site-specific incorporation of novel components into DNA, RNA, and proteins. Here, we describe the UBPs developed by three research teams and their application in PCR-based diagnostics, high-affinity DNA aptamer generation, site-specific labeling of RNAs, semi-synthetic organism creation, and unnatural-amino-acid-containing protein synthesis.

DNA Sequencing Method Including Unnatural Bases for DNA Aptamer Generation by Genetic Alphabet Expansion.

The creation of unnatural base pairs (UBPs) has rapidly advanced the genetic alphabet expansion technology of DNA, requiring a new sequencing method for UB-containing DNAs with five or more letters. The hydrophobic UBP, Ds-Px, exhibits high fidelity in PCR and has been applied to DNA aptamer generation involving Ds as a fifth base. Here, we present a sequencing method for Ds-containing DNAs, in which Ds bases are replaced with natural bases by PCR using intermediate UB substrates (replacement PCR) for conventional deep sequencing. The composition rates of the natural bases converted from Ds significantly varied, depending on the sequence contexts around Ds and two different intermediate substrates. Therefore, we made an encyclopedia of the natural-base composition rates for all sequence contexts in each replacement PCR using different intermediate substrates. The Ds positions in DNAs can be determined by comparing the natural-base composition rates in both the actual and encyclopedia data, at each position of the DNAs obtained by deep sequencing after replacement PCR. We demonstrated the sequence determination of DNA aptamers in the enriched Ds-containing DNA libraries isolated by aptamer generation procedures targeting proteins. This study also provides valuable information about the fidelity of the Ds-Px pair in replication.

Genetic Alphabet Expansion Provides Versatile Specificities and Activities of Unnatural-Base DNA Aptamers Targeting Cancer Cells

The potential of genetic alphabet expansion technologies using artificial extra base pairs (unnatural base pairs) has been rapidly expanding and increasing. We present that the hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which acts as a fifth letter in a DNA library, provides a series of high-affinity DNA aptamers with versatile binding specificities and activities to cancer cells. These Ds-containing DNA aptamers were generated by a method called cell-ExSELEX to target three breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. Aptamer 14A-MCF7, which targets MCF7 cells, specifically binds to MCF7 cells, but not other cancer cell lines. Aptamer 07-MB231, which targets MDA-MB-231 cells, binds to a series of metastatic bone and lung cancer cell lines. Aptamer 05-MB231 targets MDA-MB-231 cells, but it also binds to all of the cancer and leukemia cell lines that we examined. None of these aptamers bind to normal cell lines, such as MCF10A and HUVEC. In addition, aptamers 14A-MCF7 and 05-MB231 are internalized within the cancer cells, and aptamer 05-MB231 possesses anti-proliferative properties against most cancer cell lines that we examined. These aptamers and the generation method are broadly applicable to cancer cell imaging, biomarker discovery, cancer cell profiling, anti-cancer therapies, and drug delivery systems.

Visual Detection of Amplified DNA by Polymerase Chain Reaction Using a Genetic Alphabet Expansion System.

Visual DNA amplification using a simple polymerase chain reaction (PCR) device is useful for field tests to detect target DNA and RNA. We hereby describe a detection system involving PCR amplification visualized with the naked eye, by genetic alphabet expansion. The system employs fluorescence resonance energy transfer (FRET) between unnatural base combinations: self-quenched dinucleotides of 2-amino-6-(2-thienyl)purine (s) as a donor and Cy3-conjugated 2-nitro-4-propynylpyrrole (Cy3-hx-Px) as an acceptor. During PCR, the triphosphate substrate of Cy3-hx-Px (Cy3-hx-dPxTP) is incorporated into DNA opposite its pairing partner, 7-(2-thienyl)-imidazo[4,5- b]pyridine (Ds), in the primer, which also contains the dinucleotides of s. Thus, the amplified DNA can be visualized by the Cy3 fluorescence resulting from the FRET between the s-dinucleotides and the incorporated Cy3-hx-Px upon 365 nm irradiation. Using this system, we demonstrated the visual single nucleotide polymorphism detection of a series of quinolone-resistant bacteria genes.

Evolving Aptamers with Unnatural Base Pairs.

A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet Expansion for SELEX) method to generate Ds-containing DNA aptamers. © 2017 by John Wiley & Sons, Inc.

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

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

Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs.

Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds-Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins. Here, to further scrutinize the functions of this highly hydrophobic Ds base, the thermal stabilities of double-stranded DNAs (dsDNA) containing a noncognate Ds-Ds or G-Ds pair were examined. The thermal stability of the Ds-Ds self-pair was as high as that of the natural G-C pair, and apart from the generally higher stability of the G-C pair than that of the A-T pair, most of the 5'-pyrimidine-Ds-purine-3' sequences, such as CDsA and TDsA, exhibited higher stability than the 5'-purine-Ds-pyrimidine-3' sequences, such as GDsC and ADsC, in dsDNAs. This trait enabled the GC-content-independent control of the thermal stability of the designed dsDNA fragments. The melting temperatures of dsDNA fragments containing the Ds-Ds pair can be predicted from the nearest-neighbor parameters including the Ds base. In addition, the noncognate G-Ds pair can efficiently distinguish its neighboring cognate natural base pairs from noncognate pairs. We demonstrated that real-time PCR using primers containing Ds accurately detected a single-nucleotide mismatch in target DNAs. These unique properties of the Ds base that affect the stabilities of the neighboring base pairs could impart new functions to DNA molecules and technologies.

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

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

Genetic alphabet expansion transcription generating functional RNA molecules containing a five-letter alphabet including modified unnatural and natural base nucleotides by thermostable T7 RNA polymerase variants.

Thermostable T7 RNA polymerase variants were explored for genetic alphabet expansion transcription involving the unnatural Ds-Pa pair. One variant exhibited high incorporation efficiencies of functionally modified Pa substrates and enabled the simultaneous incorporation of 2'-fluoro-nucleoside triphosphates of pyrimidines into transcripts, allowing the generation of novel, highly functional RNA molecules.

High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases

The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165.

High Fidelity, Efficiency and Functionalization of Ds-Px Unnatural Base Pairs in PCR Amplification for a Genetic Alphabet Expansion System.

Genetic alphabet expansion of DNA using an artificial extra base pair (unnatural base pair) could augment nucleic acid and protein functionalities by increasing their components. We previously developed an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px), which exhibits high fidelity as a third base pair in PCR amplification. Here, the fidelity and efficiency of Ds-Px pairing using modified Px bases with functional groups, such as diol, azide, ethynyl and biotin, were evaluated by an improved method with optimized PCR conditions. The results revealed that all of the base pairs between Ds and either one of the modified Px bases functioned with high amplification efficiency (0.76-0.81), high selectivity (≥99.96% per doubling), and less sequence dependency, in PCR using 3'-exonuclease-proficient Deep Vent DNA polymerase. We also demonstrated that the azide-Px in PCR-amplified DNA was efficiently modified with any functional groups by copper-free click reaction. This genetic alphabet expansion system could endow nucleic acids with a wide variety of increased functionalities by the site-specific incorporation of modified Px bases at desired positions in DNA.