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Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ - :dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ - :dZ pairs join parallel strands in a four-stranded compact down-up-down-up fold. These have two possible structures: one with intercalated dZ - :dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC + -reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis., (© 2024. The Author(s).)

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition., (© 2023. The Author(s).)

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo-[1,2- a ]-1,3,5-triazin-(8 H )-4-one and 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-(1 H )-pyridin-2-one, abbreviated as P and Z . To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find G-Z pairs have stability comparable to that of A-T pairs and should therefore be included as base pairs in structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include the P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing it with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases in which Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

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

A fundamental property of DNA built from four informational nucleotide units (GCAT) is its ability to adopt different helical forms within the context of the Watson-Crick pair. Well-characterized examples include A-, B-, and Z-DNA. For this study, we created an isoinformational biomimetic polymer, built (like standard DNA) from four informational "letters", but with the building blocks being artificial. This ALternative Isoinformational ENgineered (ALIEN) DNA was hypothesized to support two nucleobase pairs, the P : Z pair matching 2-amino-imidazo-[1,2 a ]-1,3,5-triazin-[8 H ]-4-one with 6-amino-3-5-nitro-1 H -pyridin-2-one and the B : S pair matching 6-amino-4-hydroxy-5-1 H -purin-2-one with 3-methyl-6-amino-pyrimidin-2-one. We report two structures of ALIEN DNA duplexes at 1.2 Å resolution and a third at 1.65 Å. All of these are built from a single self-complementary sequence (5'-CT SZZPBSBSZPPB AG) that includes 12 consecutive ALIEN nucleotides. We characterized the helical, nucleobase pair, and dinucleotide step parameters of ALIEN DNA in these structures. In addition to showing that ALIEN pairs retain basic Watson-Crick pairing geometry, two of the ALIEN DNA structures are characterized as A-form DNA and one as B-form DNA. We identified parameters that map differences effecting the transition between the two helical forms; these same parameters distinguish helical forms of isoinformational natural DNA. Collectively, our analyses suggest that ALIEN DNA retains essential structural features of natural DNA, not only its information density and Watson-Crick pairing but also its ability to adopt two canonical forms.

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six-letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six-letter DNA library to selectively bind liver cancer cells; and 2) in a six-letter self-assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer-nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer-targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work., (Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.)

In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.

Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.

Thermal denaturation and circular dichroism studies suggested that multiple (up to 12), sequential pseudothymidines, a representative C-glycoside, do not perturb the structure of a representative DNA duplex. Further, various Family A and B DNA polymerases were found to extend a primer by incorporating four sequential pseudothymidine triphosphates, and then continue the extension to generate full-length product. Detailed studies showed that Taq polymerase incorporated up to five sequential C-glycosides, but not more. These results constrain architectures for sequencing, quantitating, and analyzing DNA analogs that exploit C-glycosides, and define better the challenge of creating a synthetic biology using these with natural polymerases.

Reported here is a "Self-Avoiding Molecular Recognition Systems" (SAMRS), a species of DNA that can bind via simple rules to natural DNA but cannot bind to other members of the same SAMRS species. A system having these properties has been achieved with 2-aminopurine-2'-deoxyriboside (A*), 2'-deoxy-2-thiothymidine (T*), 2'-deoxyinosine (G*) and N4-ethyl-2'-deoxycytidine. These were designed to form more stable base pairs with natural complements than with SAMRS complements, based on the number of hydrogen bonds. Thermal melting studies were performed using duplexes containing SAMRS components. All SAMRS species, A*, T*, G* and C*, formed more stable base pairs with natural complements, T, A, C and G than with SAMRS complements, T*, A*, C* and G* respectively. This property of SAMRS would be useful for avoiding to be produced undesired products derived from intra- and intermolecular interaction between primers in multiplexed polymerase chain reactions.

In this paper, we describe the synthesis of the 3'-3'-linked TFOs that can form the antiparallel triplexes with the duplex DNA target by reverse Hoogsteen hydrogen bonds. Stability of the alternate-strand triplexes between these TFOs and the target DNAs was investigated using the electrophoretic mobility shift assay (EMSA). It was found that the alternate-strand triplexes were significantly stabilized by linking the TFO fragments with the pentaerythritol linker. And, unlike the alternate-strand triplexes composed of the pyrimidine motif, the terminal ammonium ion of the aminobutyl-linker and the intercalator of the TFOs did not contribute to the stability of the alternate-strand triplex comprised of the purine motif. We also tested the ability of the 3'-3'-linked TFOs to inhibit cleavage of the duplex DNA target 17 by the restriction enzyme EcoT14I and found that the 3'-3'-linked TFOs 12 and 13 inhibited the cleavage by the enzyme more effectively than the unlinked decamer 8. Thus, the TFOs linked with pentaerythritol may be useful as the antigene oligonucleotide to the DNA targets, which have alternating oligopyrimidine-oligopurine sequences.

3'-3'-Linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point were synthesized on a DNA synthesizer using a controlled pore glass (CPG), which has pentaerythritol carrying the intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer. The inhibitory activity of the 3'-3'-linked ODNs against the cleavage of the target DNA by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer and the 3'-3'-linked ODN without the intercalator.