Genetic Alphabet Research Articles

Refining the Genetic Alphabet: A Late-Period Selection Pressure?

The transition from genomic ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) in primitive cells may have created a selection pressure that refined the genetic alphabet, resulting from the global weakening of the N-glycosyl bonds. Hydrolytic rupture of these bonds, termed deglycosylation, leaves an abasic site that is the single greatest threat to the stability and integrity of genomic DNA. The rates of deglycosylation are highly dependent on the identity of the nucleobases. Modifications made to the bases, such as deamination, oxidation, and alkylation, can further increase deglycosylation reaction rates, suggesting that the native bases provide optimum N-glycosyl bond stability. To protect their genomes, cells have evolved highly specific enzymes called glycosylases, associated with DNA repair, that detect and remove these damaged bases. In RNA, however, the occurrence of many of these modified bases is deliberate. The dichotomous behavior that cells exhibit toward base modifications may have originated in the RNA world. Modified bases would have been advantageous for the functional and structural repertoire of catalytic RNAs. Yet in an early DNA world, the utility of these heterocycles was greatly diminished, and their presence posed a distinct liability to the stability of cells' genomes. A natural selection for bases exhibiting the greatest resistance to deglycosylation would have ensured the viability of early DNA life, along with the recruitment of DNA repair.

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.

Interaction of RNA molecules: Binding energy and the statistical properties of random sequences

The free binding energy of two RNA molecules is determined and some statistical properties such as fluctuation of the mean binding energy between two RNA molecules and the distribution of loop lengths in the structure formed are discussed. An analysis of the dependence of the specific free energy of a complex of two long random RNA molecules on the number c of nucleotide types led us to suggest that the four-letter genetic alphabet used by nature plays a special role.

Metal-Mediated DNA Base Pairing: Alternatives to Hydrogen-Bonded Watson–Crick Base Pairs

With its capacity to store and transfer the genetic information within a sequence of monomers, DNA forms its central role in chemical evolution through replication and amplification. This elegant behavior is largely based on highly specific molecular recognition between nucleobases through the specific hydrogen bonds in the Watson-Crick base pairing system. While the native base pairs have been amazingly sophisticated through the long history of evolution, synthetic chemists have devoted considerable efforts to create alternative base pairing systems in recent decades. Most of these new systems were designed based on the shape complementarity of the pairs or the rearrangement of hydrogen-bonding patterns. We wondered whether metal coordination could serve as an alternative driving force for DNA base pairing and why hydrogen bonding was selected on Earth in the course of molecular evolution. Therefore, we envisioned an alternative design strategy: we replaced hydrogen bonding with another important scheme in biological systems, metal-coordination bonding. In this Account, we provide an overview of the chemistry of metal-mediated base pairing including basic concepts, molecular design, characteristic structures and properties, and possible applications of DNA-based molecular systems. We describe several examples of artificial metal-mediated base pairs, such as Cu(2+)-mediated hydroxypyridone base pair, H-Cu(2+)-H (where H denotes a hydroxypyridone-bearing nucleoside), developed by us and other researchers. To design the metallo-base pairs we carefully chose appropriate combinations of ligand-bearing nucleosides and metal ions. As expected from their stronger bonding through metal coordination, DNA duplexes possessing metallo-base pairs exhibited higher thermal stability than natural hydrogen-bonded DNAs. Furthermore, we could also use metal-mediated base pairs to construct or induce other high-order structures. These features could lead to metal-responsive functional DNA molecules such as artificial DNAzymes and DNA machines. In addition, the metallo-base pairing system is a powerful tool for the construction of homogeneous and heterogeneous metal arrays, which can lead to DNA-based nanomaterials such as electronic wires and magnetic devices. Recently researchers have investigated these systems as enzyme replacements, which may offer an additional contribution to chemical biology and synthetic biology through the expansion of the genetic alphabet.

Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

Synthesis and Incorporation into Oligodeoxynucleotides of Carbocyclic exo-Amino Nucleosides

The preparation of a series of carbocyclic exo-amino nucleosides from selected primary aromatic amines in both the C1′ α- and β-epimeric form as well as the corresponding building blocks for DNA synthesis is described. These nucleosides were incorporated into oligodeoxynucleotides and their base-pairing properties with natural bases and, in part, with themselves investigated. The results obtained confirm that such nucleoside analogues engage in specific interactions with natural nucleotides in DNA duplexes. In addition they can form self-pairs that match or exceed the stability of Watson-Crick base-pairs. Thus, carbocyclic exo-amino nucleosides can be taken into consideration in the design of novel base-pairs for the extension of the genetic alphabet, or for other applications in biotechnology. 1 Introduction 2 Synthetic Routes 2.1 Synthesis of Carbocyclic exo-Amino Nucleosides and Building Blocks 2.2 Synthesis of Oligonucleotides 3 Pairing Properties of Oligonucleotides 4 Discussion and Conclusions

Natural versus Artificial Creation of Base Pairs in DNA: Origin of Nucleobases from the Perspectives of Unnatural Base Pair Studies

Since life began on Earth, the four types of bases (A, G, C, and T(U)) that form two sets of base pairs have remained unchanged as the components of nucleic acids that replicate and transfer genetic information. Throughout evolution, except for the U to T modification, the four base structures have not changed. This constancy within the genetic code raises the question of how these complicated nucleotides were generated from the molecules in a primordial soup on the early Earth. At some prebiotic stage, the complementarity of base pairs might have accelerated the generation and accumulation of nucleotides or oligonucleotides. We have no clues whether one pair of nucleobases initially appeared on the early Earth during this process or a set of two base pairs appeared simultaneously. Recently, researchers have developed new artificial pairs of nucleobases (unnatural base pairs) that function alongside the natural base pairs. Some unnatural base pairs in duplex DNA can be efficiently and faithfully amplified in a polymerase chain reaction (PCR) using thermostable DNA polymerases. The addition of unnatural base pair systems could expand the genetic alphabet of DNA, thus providing a new mechanism for the generation novel biopolymers by the site-specific incorporation of functional components into nucleic acids and proteins. Furthermore, the process of unnatural base pair development might provide clues to the origin of the natural base pairs in a primordial soup on the early Earth. In this Account, we describe the development of three representative types of unnatural base pairs that function as a third pair of nucleobases in PCR and reconsider the origin of the natural nucleic acids. As researchers developing unnatural base pairs, they use repeated "proof of concept" experiments. As researchers design new base pairs, they improve the structures that function in PCR and eliminate those that do not. We expect that this process is similar to the one functioning in the chemical evolution and selection of the natural nucleobases. Interestingly, the initial structures designed by each research group were quite similar to those of the latest successful unnatural base pairs. In this regard, it is tempting to form a hypothesis that the base pairs on the primordial Earth, in which the natural purine bases, A and G, and pyrimidine bases, C and T(U), originated from structurally similar compounds, such as hypoxanthine for a purine base predecessor. Subsequently, the initial base pair evolved to the present two sets of base pairs via a keto-enol tautomerization of the initial compounds.

Expansion of the Genetic Alphabet: Unnatural Nucleobases and Their Applications

Nucleic acids are essential biomolecules that encode all of the information necessary for life. Specific pairing of A with T (or U) and C with G during replication, transcription, and translation is the key to effective transmission of genetic information between generations, as well as accurate conversion of genetic information into protein sequence. Given the magnitude of the tasks orchestrated by the Watson-Crick base pairing, it is striking to consider that biological systems accomplish these tasks using only four nucleobases. Realizing the powerful nature of base-pair recognition, researchers have been inspired to ask the question of whether the genetic code can be artificially expanded to generate biological systems having novel functions. It was this question that led Alex Rich in 1962 to propose the concept of orthogonal base pairing between iso-G and iso-C and inspired Professor Steven A. Benner in the late 1980s to expand the genetic alphabet from four to six letters. Benner's early research focused on the development of new base pairs having hydrogen bonding patterns orthogonal to those in the canonical Watson-Crick base pairs. In 1994, Professor Eric T. Kool opened a new functional dimension with the creation of nonhydrogen bonding unnatural nucleobase surrogates. Expansion of the genetic alphabet has dramatically increased the functional potential of DNA, for example, by enabling site-directed oligonucleotide labeling and in vitro selections with oligonucleotides having increased chemical diversity. Translation of an expanded DNA alphabet into RNA is a challenging task, but one which has potential to give rise to semisynthetic organisms with increased biodiversity. This special issue highlights recent accomplishments at the interface of organic chemistry and molecular biology which hold promise to further expand the potential of nucleic acids having unnatural nucleobases. Specifically, the reports in this special issue focus on the synthesis of unnatural nucleobases and nucleic acid backbones, the exploration of their structure and duplex stabilizing ability, and the polymerase mediated replication and transcription of DNA containing unnatural nucleobases. T. Lonnberg and a coworker report the synthesis and study of a bis(pyrazolyl)purine ribonucleoside having increased hydrophobic surface area and the ability to form complex with metal ions. The hydrogen bonding pattern of this nucleoside makes it complementary to thymine and uridine. The authors demonstrate that the bis(pyrazolyl) nucleobase is capable of forming a Pd2+-mediated base pair with uridine in the monomeric state. When incorporated into an oligonucleotide, the bis(pyrazolyl) nucleobase stabilizes DNA duplexes when paired with thymine, but this stabilization appears to result from increased π-stacking interactions rather than metal complexation. These studies open the door to applications using unnatural nucleobases to increase the binding affinity of probes and therapeutics targeted at native DNA and RNA. Much effort has focused on the incorporation of unnatural nucleobases into native DNA and RNA, and the availability of nonnative backbones such as LNA, PNA, and GNA opens the door for further expansion of nucleic acid structure and function. C. Forster and coworkers evaluate the structural properties of “all LNA” duplexes and demonstrate that these duplexes have a relaxed helical structure unique from that of DNA and RNA. J. M. Heemstra and coworkers report an expedient synthetic route to the Fmoc-protected PNA backbone, which serves as a key intermediate in the synthesis of PNA monomers having unnatural nucleobases. Together, these studies facilitate the exploration of nucleic acids having both nonnative backbones and unnatural nucleobases, which is in turn anticipated to provide molecules having novel structural and functional properties. The ability to use native or engineered replication and transcription machinery with nucleic acids containing unnatural nucleobases is critical to many in vitro and in vivo applications. I. Hirao and coworkers describe a two-unnatural-base-pair system capable of incorporating unnatural nucleobases into DNA via polymerase chain reaction, then into RNA via T7 transcription. Using this system, they demonstrate sequence-specific incorporation of a biotinylated nucleobase into a 260-mer RNA sequence. Generating modified RNA of this length is not feasible using purely synthetic methods; thus this work significantly improves access to site-specifically labeled RNA sequences. Additionally, P. J. Beuning and a coworker review the use of native A and Y family DNA polymerases for replication of DNA containing unnatural nucleobases. This review serves as an excellent resource for researchers seeking to utilize unnatural nucleobases and provides significant insight into how polymerases deal with both synthetic and damaged DNA nucleobases. Collectively, these reports demonstrate the potential of unnatural nucleobases for applications in chemistry and biology and offer valuable tools for further exploring nucleic acid structural diversity.

PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds-Px and Ds-Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma.

Toward the expansion of the genetic alphabet of DNA, several artificial third base pairs (unnatural base pairs) have been created. Synthetic DNAs containing the unnatural base pairs can be amplified faithfully by PCR, along with the natural A–T and G–C pairs, and transcribed into RNA. The unnatural base pair systems now have high potential to open the door to next generation biotechnology. The creation of unnatural base pairs is a consequence of repeating “proof of concept” experiments. In the process, initially designed base pairs were modified to address their weak points. Some of them were artificially evolved to ones with higher efficiency and selectivity in polymerase reactions, while others were eliminated from the analysis. Here, we describe the process of unnatural base pair development, as well as the tests of their applications.

Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry

Toward new biotechnology by genetic alphabet expansion, we developed an efficient site-specific labeling method for large RNA molecules. The combination of unnatural base pair transcription and post-transcriptional modification by click chemistry enables simple RNA labeling with a wide variety of functional groups at desired positions, in a one-pot reaction.

Classical and "Quantum-like" Views of the Genetic Code

We are very pleased to present this Special Issue of NeuroQuantology Journal, devoted to the genetic code and related topics, and kindly made possible by Sultan Tarlaci, the Editor-In-Chief. The origin and the structure of the genetic code of Earth’s living organisms is still a big challenge for Science, with many unanswered questions as for example its “degeneracy”. Since about two decades, its study has attracted many researchers throughout the World, chemists, physicists, mathematicians and others with extremely various and prolific approaches. The presented collection of papers, in this Special Issue, constitutes certainly only a “snapshot” of recent and important researches in this field and made by quality researchers who have very kindly accepted to contribute. It is also a good place, here, to acknowledge the actions of Sultan Tarlaci, in the dissemination of new ideas and to address him all my thanks and, on their behalf, those of my guest authors. We begin with the review by Miloje Rakocevic who explains us, beautifully, that his very long investigation shows that the genetic code is indeed a coherent system. This also a good place to acknowledge his constant help and advice, and also his great disponibility concerning the present Special Issue. I take, also here, the opportunity to address him, once more, all my wishes at the occasion of his 73th birthday, in this year 2011. Next, we have a series of original research papers. Branko Dragovich review his recent researches and presents some new results on the p-adic structure of the genetic code. The present author, Tidjani Negadi, contributes three papers where he uses several simple arithmetic functions, as useful tools, to investigate the physico-mathematical structure of the genetic code, with several new and amazing results. In particular our “quantum-like” approach of the genetic code describes nicely the encasing and the inter-relationships of many results obtained by Rakocevic. Natasa Misic proposes us a nice paper where she shows that famous shCherbak’s Prime Quantum 037 has nested, numeric, geometric and arithmetic properties and that it could constitute a basis for biological coding/computing. Also, Castro-Chavez, in a first paper, compares the genome of Man with the genome of other organisms by using the “rotating circular genetic code” and, in a second paper; he transfers the classic “rotating circular genetic” code to the “64-grid model”. Sergey Petoukhov presents a review of his recent work on matrix genetics and the algebraic properties of the multi‐level system of genetic alphabets. Finally, the work by Diego Lucio Rapoport could be best described, using words, by Professor Stein Erik Johansen (Dragvoll, Norway) saying that Diego Rapoport “has achieved plural novel results of great importance during the last three years, involving pieces of knowledge from many scientific fields becoming integrated in his Kleinbottle paradigm…”; “…he has had the mathematical competence and originality to convert Kleinbottle topology into important extensions of matrix logic with related informatics and cybernetics, into integration with torsion physics previously developed much by himself, and in recent times also with radical novel implications for genetics, chemistry and segments of neuroscience.” It is well understood that the presented (quality) works and views are those of individual authors, and would not be necessarily shared by the present guest Editor, the Chief-Editor or the Board of NeuroQuantology Journal. Dr. Tidjani Negadi Universite d’Oran Theme Issue, Guest Editor

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds–Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds–Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds–Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds–Px pair system provides a framework for new biotechnology.

Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs.

Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.

Matrix Genetics and Algebraic Properties of the Multi-Level System of Genetic Alphabets

The article is devoted to algebraic properties of the multi-level system of molecular-genetic alphabets. It leads to help solve the problem of algebraic unity of inherited information systems in living matter. These algebraic properties are revealed by means of Kronecker families of matrix forms of a presentation of molecular-genetic alphabets. A family of genetic (8x8)-matrices shows unexpected connections of the genetic system with Rademacher and Walsh functions and with special Hadamard matrices which are well-known in theory of noise-immunity coding and digital communication. Decompositions of such genetic (8x8)-matrices on the basis of the known principle of dyadic-shifts lead to sets of 8 sparse matrices. Each of these sets is closed in relation to multiplication and defines a special algebra of 8-dimensional hypercomplex numbers. Mathematical aspects of these 8-dimensional algebras are presented in connection with metric vector spaces, the sequency theory by Harmuth and some methods of spectral analysis. The diversity of known dialects of the genetic code can be analyzed from the viewpoint of these algebras. Our results are discussed taking into account the important role of dyadic shifts, hypercomplex numbers and Hadamard matrices in mathematics, informatics, theoretical physics, etc. These results testify that living matter has a profound algebraic essence which is interconnected with 8-dimensional vector spaces. In our opinion these results lead to a new way of knowledge of living matter in the field of algebraic biology and its mathematical modeling. The idea of a biological meaning of Kronecker multiplication of matrices is based on the structure of Punnett squares in the field of Mendelian genetics. The author believes that the Mendelian laws of independent inheritance of traits have revealed just the tip of an algebraic iceberg of informational structure of living matter and that matrix genetics has contributed to the next steps to disclose this important iceberg.

Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair

The universal genetic code relies on two hydrogen-bonded Watson-Crick base pairs that can form 64 triplet codons. This places a limit on the number of amino acids that can be encoded, which has motivated efforts to create synthetic base pairs that are orthogonal to the natural ones. An additional base pair would result in another 61 triplet codons. Artificial organic base pairs have been described in enzymatic incorporation studies, and inorganic T-Hg-T and C-Ag-C base pairs have been reported to form in primer extension studies. Here, we demonstrate a metal base pair that is fully orthogonal and can be replicated, and can even be amplified by polymerase chain reaction in the presence of the canonical pairs dA:dT and dG:dC. Crystal structures of a dS-Cu-dS base pair inside a polymerase show that reversible chemistry is possible directly inside the polymerase, which enables the efficient copying of the inorganic crosslink. The results open up the possibility of replicating and amplifying artificial inorganic DNA nanostructures by extending the genetic alphabet.

What exactly is synthetic biology?

Summary Having spent the 20 th century taking living things apart, biologists are now beginning to put pieces together in new forms. Synthetic biology emerges as a new discipline with a few success stories, but no clear direction yet. Michael Gross reports.

Unnatural base pair systems for sensing and diagnostic applications

Expansion of the genetic alphabet by an unnatural base pair system provides a platform for the site-specific, enzymatic incorporation of extra, functional components into nucleic acids. Recently, several unnatural base pairs that exhibit high fidelity and efficiency in PCR have been developed. Functional groups of interest, such as fluorescent dyes, can be linked to the unnatural bases, and the modified base substrates are site-specifically incorporated into nucleic acids by polymerases. Furthermore, unique unnatural base pairs between fluorophore and quencher base analogs have been developed for imaging PCR amplification and as molecular beacons. Here, we describe the recent progress in the development of unnatural base pairs that function in PCR amplification and their applications as sensing and diagnostic tools.

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor–donor–acceptor and acceptor–acceptor–donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells.