Genetic Alphabet Research Articles

Electrodeposition of silver amalgam particles on screen-printed silver electrodes in voltammetric detection of 4-nitrophenol, bovine serum albumin and artificial nucleosides dTPT3 and d5SICS

Electrodeposition of silver amalgam particles (AgAPs) on various substrates represents a prospective approach to the development of a novel detection system applicable in the study of various electrochemically active substances, including nucleic acids and proteins. Herein, a double pulse chronoamperometric deposition of AgAPs on in-house fabricated screen-printed silver electrodes (SPAgE) has been optimized using the voltammetric signal of a model electrochemically reducible organic nitro-compound, 4-nitrophenol (4-NP). The surface morphology of SPAgE-AgAP was monitored by scanning electron microscopy with an energy dispersive X-ray spectrometer. A compact three-electrode sensor consisting of the working (substrate) SPAgE, counter graphite, and pseudo-reference Ag|AgCl electrodes was designed for analysis of electrochemically reducible compounds in a 96-well plate with about 150-μl sample volume per well. Herein optimized SPAgE-AgAP allowed detection of 4-NP down to 1 μmol l−1 using cyclic voltammetry. Advantageously, differential pulse voltammetry at SPAgE-AgAP allowed highly sensitive detection of artificial nucleosides dTPT3 and d5SICS (developed to expand genetic alphabet in semi-synthetic organisms) using their catalytic hydrogen evolution signals, with limits of detection of 0.4 pmol l−1. Moreover, these artificial nucleosides were easily detectable in the excess of natural nucleosides down to molar ratio 1:12000. Using constant current chronopotentiometric stripping and the catalytic peak H, we further demonstrated detection of a protein, bovine serum albumin, at the SPAgE-AgAP. Thus, we present the SPAgE-AgAP as a potent tool applicable in simple, fast, and sensitive electrochemical detection of reducible or catalytically active species, with prospective applications in hot research areas including chemical and synthetic biology.

Robustness against point mutations of genetic code extensions under consideration of wobble-like effects

Many theories of the evolution of the genetic code assume that the genetic code has always evolved in the direction of increasing the supply of amino acids to be encoded (Barbieri, 2019; Di Giulio, 2005; Wong, 1975). In order to reduce the risk of the formation of a non-functional protein due to point mutations, nature is said to have built in control mechanisms. Using graph theory the authors have investigated in Blazej et al. (2019) if this robustness is optimal in the sense that a different codon–amino acid assignment would not generate a code that is even more robust. At present, efforts to expand the genetic code are very relevant in biotechnological applications, for example, for the synthesis of new drugs (Anderson et al., 2004; Chin, 2017; Dien et al., 2018; Kimoto et al., 2009; Neumann et al., 2010). In this paper we generalize the approach proposed in Blazej et al. (2019) and will explore hypothetical extensions of the standard genetic code with respect to their optimal robustness in two ways:(1) We keep the usual genetic alphabet but move from codons to longer words, such as tetranucleotides. This increases the supply of coding words and thus makes it possible to encode non-canonical amino acids.(2) We expand the genetic alphabet by introducing non-canonical base pairs. In addition, the approach from Blazej et al. (2019) and Blazej et al. (2018) is extended by incorporating the weights of single point-mutations into the model. The weights can be interpreted as probabilities (appropriately normalized) or degrees of severity of a single point mutation. In particular, this new approach allows us to take a closer look at the wobble effects in the translation of codons into amino acids. According to the results from Blazej et al. (2019) and Blazej et al. (2018), the standard genetic code is not optimal in terms of its robustness to point mutations if the weights of single point mutations are not taken into account. After incorporation into the model weights that mimic the wobble effect, the results of the present work show that it is much more robust, almost optimal in that respect.We hope, that this theoretical analysis might help to assess extended genetic codes and their abilities to encode new amino acids.

Strategies for Covalent Labeling of Long RNAs.

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site‐specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.

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.

Transcription and Reverse Transcription of an Expanded Genetic Alphabet In Vitro and in a Semisynthetic Organism.

Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

Electrochemical reduction and oxidation of eight unnatural 2′-deoxynucleosides at a pyrolytic graphite electrode

Recently we showed the reduction and oxidation of six natural 2′-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2′-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding.

Th formation of metal base pairs is a versatile method for the introduction of metal cations into nucleic acids that has been used in numerous applications including the construction of metal nanowires, development of energy, charge-transfer devices and expansion of the genetic alphabet. As an alternative, enzymatic construction of metal base pairs is an alluring strategy that grants access to longer sequences and offers the possibility of using such unnatural base pairs (UBPs) in SELEX experiments for the identification of functional nucleic acids. This method remains rather underexplored, and a better understanding of the key parameters in the design of efficient nucleotides is required. We have investigated the effect of methylation of the imidazole nucleoside (dImnMe TP) on the efficiency of the enzymatic construction of metal base pairs. The presence of methyl substituents on dImTP facilitates the polymerase-driven formation of dIm4Me -AgI -dIm and dIm2Me TP-CrIII -dIm base pairs. Steric factors rather than the basicity of the imidazole nucleobase appear to govern the enzymatic formation of such metal base pairs. We also demonstrate the compatibility of other metal cations rarely considered in the construction of artificial metal bases by enzymatic DNA synthesis under both primer extension reaction and PCR conditions. These findings open up new directions for the design of nucleotide analogues for the development of metal base pairs.

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

Conformational Conversion Enhances Cellular Uptake of F Base Double-Strand-Conjugated Oligonucleotides.

Artificial bases have emerged as a useful tool to expand genetic alphabets and biomedical applications of oligonucleotides. Herein, we reported that the conformation conversion enhances cellular uptake of hydrophobic 3,5-bis(trifluoromethyl)benzene (F) base double-strand-conjugated oligonucleotides. The formation of the F base double-strand caged the hydrophobic F base in the duplex strand, thus preventing F base from interacting with cells to some extent. However, upon conversion of F base double-strand-conjugated oligonucleotide to F base single-strand-conjugated oligonucleotide, F bases then were allowed to interact with cells by stronger hydrophobic interactions, followed by cellular uptake. The results were concluded as a pairing-induced cage effect of F base and have the potential for the construction of stimuli-responsive cellular uptake of functional nucleic acids.

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.

New codons for efficient production of unnatural proteins in a semisynthetic organism.

Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 pair to form an unnatural base pair (UBP) and allow for the creation of semi-synthetic organisms (SSOs) with additional sense codons. Here we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded non-canonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to site-specifically incorporate multiple, different ncAAs into a protein should now allow for the development of proteins with novel activities and possibly even SSOs with new forms and functions.

Enzymatic synthesis of biphenyl-DNA oligonucleotides

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solid-phase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdT-mediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3′-end.

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.

Tautomeric Equilibria of Nucleobases in the Hachimoji Expanded Genetic Alphabet.

Evolution has yielded biopolymers that are constructed from exactly four building blocks and are able to support Darwinian evolution. Synthetic biology aims to extend this alphabet, and we recently showed that 8-letter (hachimoji) DNA can support rule-based information encoding. One source of replicative error in non-natural DNA-like systems, however, is the occurrence of alternative tautomeric forms, which pair differently. Unfortunately, little is known about how structural modifications impact free-energy differences between tautomers of the non-natural nucleobases used in the hachimoji expanded genetic alphabet. Determining experimental tautomer ratios is technically difficult, and so, strategies for improving hachimoji DNA replication efficiency will benefit from accurate computational predictions of equilibrium tautomeric ratios. We now report that high-level quantum-chemical calculations in aqueous solution by the embedded cluster reference interaction site model, benchmarked against free-energy molecular simulations for solvation thermodynamics, provide useful quantitative information on the tautomer ratios of both Watson-Crick and hachimoji nucleobases. In agreement with previous computational studies, all four Watson-Crick nucleobases adopt essentially only one tautomer in water. This is not the case, however, for non-natural nucleobases and their analogues. For example, although the enols of isoguanine and a series of related purines are not populated in water, these heterocycles possess N1-H and N3-H keto tautomers that are similar in energy, thereby adversely impacting accurate nucleobase pairing. These robust computational strategies offer a firm basis for improving experimental measurements of tautomeric ratios, which are currently limited to studying molecules that exist only as two tautomers in solution.

Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with noncanonical amino acids.

Partition Markov Model for Covid-19 Virus

In this paper, we investigate a specific structure within the theoretical framework of Partition Markov Models (PMM) [see García Jesús and González-López, Entropy 19, 160 (2017)]. The structure of interest lies in the formulation of the underlying partition, which defines the process, in which, in addition to a finite memory o associated with the process, a parameter G is introduced, allowing an extra dependence on the past complementing the dependence given by the usual memory o. We show, by simulations, how algorithms designed for the classic version of the PMM can have difficulties in recovering the structure investigated here. This specific structure is efficient for modeling a complete genome sequence, coming from the newly decoded Coronavirus Covid-19 in humans [see Wu et al., Nature 579, 265–269 (2020)]. The sequence profile is represented by 13 units (parts of the state space’s partition), for each of the 13 units, their respective transition probabilities are computed for any element of the genetic alphabet. Also, the structure proposed here allows us to develop a comparison study with other genomic sequences of Coronavirus, collected in the last 25 years, through which we conclude that Covid-19 is shown next to SARS-like Coronaviruses (SL-CoVs) from bats specimens in Zhoushan [see Hu et al., Emerg Microb Infect 7, 1–10 (2018)].

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.

Can modified DNA base pairs with chalcogen bonding expand the genetic alphabet? A combined quantum chemical and molecular dynamics simulation study.

A comprehensive (DFT and MD) computational study is presented with the goal to design and analyze model chalcogen-bonded modified nucleobase pairs that replace one (i.e., AXY:T, G:CXY, GXY:C) or two (GXY:CX'Y', X/X' = S, Se and Y/Y' = F, Cl, Br) Watson-Crick (WC) hydrogen bonds of the canonical A:T or G:C pair with chalcogen bond(s). DFT calculations on 18 base pair combinations that replace one WC hydrogen bond with a chalcogen bond reveal that the bases favorably interact in the gas phase (binding strengths up to -140 kJ mol-1) and water (up to -85 kJ mol-1). Although the remaining hydrogen bond(s) exhibits similar characteristics to those in the canonical base pairs, the structural features of the (Y-XO) chalcogen bond(s) change significantly with the identity of X and Y. The 36 doubly-substituted (GXY:CX'Y') base pairs have structural deviations from canonical G:C similar to those of the singly-substituted modifications (G:CXY or GXY:C). Furthermore, despite the replacement of two strong hydrogen bonds with chalcogen bonds, some GXY:CX'Y' pairs possess comparable binding energies (up to -132 kJ mol-1 in the gas phase and up to -92 kJ mol-1 in water) to the most stable G:CXY or GXY:C pairs, as well as canonical G:C. More importantly, G:C-modified pairs containing X = Se (high polarizability) and Y = F (high electronegativity) are the most stable, with comparable or slightly larger (by up to 13 kJ mol-1) binding energies than G:C. Further characterization of the chalcogen bonding in all modified base pairs (AIM, NBO and NCI analyses) reveals that the differences in the binding energies of modified base pairs are mainly dictated by the differences in the strengths of their chalcogen bonds. Finally, MD simulations on DNA oligonucleotides containing the most stable chalcogen-bonded base pair from each of the four classifications (AXY:T, G:CXY, GXY:C and GXY:CX'Y') reveal that the singly-modified G:C pairs best retain the local helical structure and pairing stability to a greater extent than the modified A:T pair. Overall, our study identifies two (G:CSeF and GSeF:C) promising pairs that retain chalcogen bonding in DNA and should be synthesized and further explored in terms of their potential to expand the genetic alphabet.