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

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

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

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

Strict Interactions of Fifth Letters, Hydrophobic Unnatural Bases, in XenoAptamers with Target Proteins.

XenoAptamers are DNA fragments containing additional letters (unnatural bases, UBs) that bind specifically to their target proteins with high affinities (sub-nanomolar KD values). One of the UBs is the highly hydrophobic 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which significantly increases XenoAptamers' affinities to targets. Originally, Ds was developed as a third base pair with a complementary UB, 2-nitro-4-propynylpyrrole (Px), for replication, and thus it can be used for aptamer generation by an evolutional engineering method involving PCR amplification. However, it is unclear whether the Ds base is the best component as the hydrophobic fifth-letter ligand for interactions with target proteins. To optimize the ligand structure of the fifth letter, we prepared 13 Ds variants and examined the affinities of XenoAptamers containing these variants to target proteins. The results obtained using four XenoAptamers prepared by the replacement of Ds bases with variants indicated that subtle changes in the chemical structure of Ds significantly affect the XenoAptamer affinities. Among the variants, placing either 4-(2-thienyl)pyrrolo[2,3-b]pyridine (Ys) or 4-(2-thienyl)benzimidazole (Bs) at specific Ds positions in each original XenoAptamer greatly improved their affinities to targets. The Ys and Bs bases are variants derived by replacing only one nitrogen with a carbon in the Ds base. These results demonstrate the strict intramolecular interactions, which are not simple hydrophobic contacts between UBs and targets, thus providing a method to mature XenoAptamers' affinities to targets.

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'.

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase.

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

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.

Uptake mechanisms of cell-internalizing nucleic acid aptamers for applications as pharmacological agents.

Nucleic acid aptamers, also regarded as chemical antibodies, show potential as targeted therapeutic and delivery agents since they possess unique advantages over antibodies. Generated by an iterative selection and amplification process from oligonucleotide libraries using cultured cells, the aptamers bind to their target molecules expressed on the cell surface. Excitingly, most aptamers also demonstrate a cell-internalizing property in native living cells, allowing them to directly enter the cells via endocytosis depending on the target. In this review, we discuss selection methods in generating cell-internalizing aptamers via a cell-based selection process, along with their challenges and optimization strategies. We highlight the cellular uptake routes adopted by the aptamers and also their intracellular fate after the uptake, to give an overview of their mechanism of action for applications as promising pharmacological agents.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers.

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification.

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (KD: 27-182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69-80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans' clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.

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.

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.

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.

Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA.

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10-9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers' applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.

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.

Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology.

Artificial extra base pairs (unnatural base pairs, UBPs) expand the genetic alphabet of DNA, thus broadening entire biological systems in the central dogma. UBPs function as third base pairs in replication, transcription, and/or translation, and have created a new research area, synthetic xenobiology, providing genetic engineering tools to generate novel DNAs, RNAs, and proteins with increased functionalities. Several UBPs have been developed and applied to PCR technology, DNA aptamer generation, and semi-synthetic organism creation. Among them, we developed a series of UBPs and demonstrated unique quantitative PCR and high-affinity DNA aptamer generation methods.

DNA aptamer generation by ExSELEX using genetic alphabet expansion with a mini-hairpin DNA stabilization method.

A novel aptamer generation method to greatly augment the affinity and stability of DNA aptamers was developed by genetic alphabet expansion combined with mini-hairpin DNA technology. The genetic alphabet expansion increases the physicochemical and structural diversities of DNA aptamers by introducing extra components, unnatural bases, as a fifth base, allowing for the enhancement of DNA aptamer affinities. Furthermore, the mini-hairpin DNA technology stabilizes DNA aptamers against nuclease digestion and thermal denaturation, by introducing an extraordinarily stable mini-hairpin DNA containing a GCGAAGC sequence. This novel method provides stabilized high-affinity DNA aptamers for diagnostic and therapeutic applications.

Genetic alphabet expansion biotechnology by creating unnatural base pairs.

Recent studies have made it possible to expand the genetic alphabet of DNA, which is originally composed of the four-letter alphabet with A-T and G-C pairs, by introducing an unnatural base pair (UBP). Several types of UBPs function as a third base pair in replication, transcription, and/or translation. Through the UBP formation, new components with different physicochemical properties from those of the natural ones can be introduced into nucleic acids and proteins site-specifically, providing their increased functionalities. Here, we describe the genetic alphabet expansion technology by focusing on three types of UBPs, which were recently applied to the creations of DNA aptamers that bind to proteins and cells and semi-synthetic organisms containing DNAs with a six-letter alphabet.