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A chemical approach to synthetic biology allows researchers to build new chemical monomers and evaluate their activity in a biochemical and biological context. Here we describe the work our laboratory has done in developing alternative bases for DNA, including non-hydrogen bonding base analogs, which have been used to study stacking, hydrogen bonding, and steric requirements in duplex stability and enzyme recognition; and size-expanded DNA bases for the development of a non-Watson-Crick genetic set. The designed molecules are useful in basic science, allowing a better understanding of the functions of natural DNA and RNA, and are also contributing practical new tools for biology and medicine.

A conversation with the uninitiated about work on “expanded DNA” and a “new genetic system” can lead to worried looks and a voiced concern that alien life forms will soon be unleashed from the laboratory. While this is far from true at the moment, synthetic biology does involve both the generation of new functionality in living systems, and the quest to understand and recreate life from its simplest building blocks.

The field of synthetic biology is pursued along two paths: an engineering “top down” approach, which views existing biological components as modules that can be combined in new ways to obtain new functionalities,1–3  and a chemical “bottom up” approach, which aims to understand the functioning of biological components by creating new components and testing them in a biochemical or biological context.1,4,5  In some respects these approaches have seemingly opposite goals, as chemists first want to replicate the basic processes of living systems with designed components, while engineers are using existing components to generate new activities. However, both use synthesis as a strategy to address a “grand challenge” which will test underlying theories and knowledge of chemical principles and biological systems.4 

Some of the challenges taken up through the “top-down” approach of the biological engineering community include the creation of synthetic genetic circuits,6,7  metabolic engineering for drug8,9  and fuel10,11  production, and the development of the first cell with an entirely artificial genome.12  This work has been extensively reviewed3,13–15  and is discussed in other chapters of this book.

Chemists, on the other hand, have identified the molecular components of cells and attempted to replace or alter these components while retaining their functionality. Using this “bottom-up” approach, work has included exploration of alternatives to the (deoxy)ribose-phosphate backbone found in natural genetic material,16,17  attempts to design proteins,18  and the creation of polymer-based analogs of cell membranes.19  Another goal – the topic of this chapter – is the synthesis of functional nucleic acid bases for a new or expanded genetic set.20 

Designing new bases and base pairs and studying them in a biochemical context allows us to both better understand the existing genetic alphabet and expand the capabilities of the genetic code. In the short term, this has already led to the development of new tools for biotechnology21  and new probes for biological mechanisms.22  For example, one of the first novel base pairs, Benner's isoG-isoC, has been used to increase the specificity of clinical detection of HIV viral loads.23  The non-hydrogen-bonding shape mimics developed in our own laboratory can serve as tools to separate the importance of hydrogen-bonding and solvation from steric effects in biochemistry.22 

A long-term goal of this field is to develop evolving, replicating, living systems with modified genetic structures. Several groups are working toward this goal, and there has been progress including new base pairs that function in PCR,24–26  the evolution of enzymes to tolerate unnatural base pairs,27,28  the establishment of function in living cells,29–31  and the incorporation of unnatural amino acids using codons with designer bases.32,33 

In our laboratory, there have been two main design strategies. First, the study of non-polar base analogs of different shapes and sizes has allowed us to tease apart effects of shape and size in base-pairing, enzymatic recognition, and helix stability.34  This work paved the way for the later development of non-hydrogen-bonding base pairs that are used very successfully today.24,35  Second, we are working toward a genetic set that retains natural hydrogen-bonding patterns but is of expanded size.36  This genetic set is different than those mentioned above in that it is not designed to function in the context of natural DNA. Advantages of this expanded genetic set include an 8-letter genetic alphabet, inherent fluorescence, and increased duplex stability; however, it pushes the limits of what natural enzymes can tolerate. This chapter will describe both of these aspects of our research in synthetic biology, providing insight into the challenges and knowledge that synthetic nucleic acids research can bring.

In order to make working alternatives and modifications to nature's genetic set, one needs to develop an understanding of the parameters for successful design. One crucial strategy for doing this is through perturbation. By redesigning one element of the system and then determining what difference the perturbation has made, we can find out which elements are essential and where there is room for change.

In this respect, each of the three components of nucleic acids – the phosphate backbone, the ribose or deoxyribose sugar, and the nitrogenous bases – have generally been considered separately. In each case, modifications have helped to identify what the critical factors are. For example, in the quest to make antisense drugs, or drugs that would pair with complementary RNA, preventing its translation,37  the importance of the repeating charge of the phosphate backbone in stabilizing helical structure and preventing aggregation was established.38  Likewise, sugar modifications have shown that more flexible linkers tend to destabilize the duplex,39  while more rigid structures can have a stabilizing effect.40 

With regard to the nucleobases, hydrogen bonds were originally regarded to be of critical importance. Benner and coworkers reported novel nucleobases in which hydrogen-bonding arrangements alternative to those found in natural bases maintained duplex stability41  and could be recognized by DNA and RNA polymerases.41,42  On the other hand, nucleotides with blocked or deleted hydrogen-bonding groups generally destabilized the DNA duplex,43–50  and early polymerase studies showed lowered fidelity.49,51–53  However, most of these modified nucleotides varied considerably in structure as well as in hydrogen-bonding ability from their natural counterparts, making it impossible to separate steric effects from electrostatic interactions. Therefore, in the early 1990s, there were still crucial questions to be answered: How important is hydrogen-bonding in stabilizing base pairs? What are the contributions of base stacking and shape complementarity to duplex stability and enzyme recognition?

In order to start to tease apart the answers to these important questions, a new set of base analogs was needed to separate stacking and steric effects from hydrogen-bonding. A novel set of nucleosides was envisioned that would mimic the size and shape of the natural bases as closely as possible, but replace all polar functional groups with nonpolar ones (Fig. 1).54  In these analogs, referred to as “bases” in analogy to their natural counterparts, hydrogen-bond donors (N-H) are replaced with nonpolar methine or methyl groups (C-H); carbonyl groups are replaced with C-F.

Figure 1.1

Nonpolar analogs of natural nucleosides. A) Molecular structure of natural and nonpolar nucleosides; B) Space-filling models comparing the sizes, shapes and surface potentials for T vs. F and A vs. Z (bases only).

Figure 1.1

Nonpolar analogs of natural nucleosides. A) Molecular structure of natural and nonpolar nucleosides; B) Space-filling models comparing the sizes, shapes and surface potentials for T vs. F and A vs. Z (bases only).

Close modal

Analogs dF and dH, mimics for dT and dG respectively, are near-perfect shape matches, as supported by molecular modeling calculations and crystal structures.54–56  dZ and dH replace an aromatic nitrogen in dA and dC with an aromatic methine, making these two bases slightly more bulky at the base-pairing edge than their natural counterparts. However, both dF and dZ show sugar conformation and base orientations similar to those of dT and dA based on NMR data.22,55 

Although fluorocarbons are hydrophobic, an isolated C-F bond retains some polarity. Therefore, the ability of 2,4-difluorotoluene (F) to form hydrogen bonds was evaluated using NMR titration studies and 9-ethyladenine as a potential hydrogen-bonding partner.57  Results showed that while uracil and 9-ethyladenine clearly formed a complex in chloroform, no significant shift of 9-ethyladenine protons occurred in the presence of F, indicating that if any interaction is present, it is very weak. These results have been supported by additional studies, including theoretical and experimental work, suggesting that a possible F-A interaction should be much smaller in energy than a T-A interaction.58,59 

How would incorporation of these base analogs affect DNA duplex stability? To answer this question, dF, dZ, and dH were incorporated into the center of a short DNA oligomer, and hybridization studies were carried out pairing the analogs opposite a natural base or dF (Table 1).56,57,60  When F, Z, or H were paired with a natural base, there was a decrease in stability relative to the comparison A-T pair of 4–6 kcal/mol, destabilizing the duplex more than any of the comparison mismatches between T and a natural base. Furthermore, the nonpolar bases showed little discrimination among the natural bases, and the small differences that were observed did not match what would be expected based on shape complementarity. For example, Z showed a slight preference for pairing with A,60  while H showed the least destabilization when paired with G.56  However, when the nonpolar bases were paired with one another, some of the loss in duplex stability was regained; hydrophobic-hydrophobic base pairs had 1–2 kcal/mol increased stability over hydrophobic-hydrophilic base pairs, comparable to a T-T mismatch. These hydrophobic pairs still appeared to show little preference for shape. F-F, F-Z, and F-H pairs all are very similar in their effects on duplex stability.

Table 1.1

Melting temperatures for DNA duplexes containing natural mismatches or nucleoside isosteres F, Z, or H.

Base Pair X-YaTm [°C]ΔTm [°C]−ΔG°25 [kcal/mol]
T-Ab 39.4 − 12.3 
T-C 26.4 13.0 8.7 
T-G 30.7 8.7 9.3 
T-T 27.1 12.3 8.9 
F-Ab 21.4 18.0 7.4 
F-C 25 14.4 8.2 
F-G 23 16.4 8.0 
F-T 20.2 19.2 7.3 
F-F 28.6 10.8 8.9 
A-Tc 39.8 − 12.4 
Z-A 25.3 14.5 8.0 
Z-C 24.4 15.4 7.9 
Z-G 23.8 16.0 7.6 
Z-T 20.8 19.0 7.5 
Z-F 30.3 9.5 8.9 
A-Hd 28.5 11.3 6.7 
C-H 28.2 11.6 6.5 
G-H 30.0 9.8 6.9 
T-H 27.6 12.2 6.6 
F-H 33.7 6.1 7.6 
T-G 36.7 3.1 8.3 
Base Pair X-YaTm [°C]ΔTm [°C]−ΔG°25 [kcal/mol]
T-Ab 39.4 − 12.3 
T-C 26.4 13.0 8.7 
T-G 30.7 8.7 9.3 
T-T 27.1 12.3 8.9 
F-Ab 21.4 18.0 7.4 
F-C 25 14.4 8.2 
F-G 23 16.4 8.0 
F-T 20.2 19.2 7.3 
F-F 28.6 10.8 8.9 
A-Tc 39.8 − 12.4 
Z-A 25.3 14.5 8.0 
Z-C 24.4 15.4 7.9 
Z-G 23.8 16.0 7.6 
Z-T 20.8 19.0 7.5 
Z-F 30.3 9.5 8.9 
A-Hd 28.5 11.3 6.7 
C-H 28.2 11.6 6.5 
G-H 30.0 9.8 6.9 
T-H 27.6 12.2 6.6 
F-H 33.7 6.1 7.6 
T-G 36.7 3.1 8.3 
a

Measured in the sequence d(CTTTTCXTTCTT)·d(AAGAAYGAAAAG) in 100 mM NaCl, 10 mM MgCl2, 10 mM Na·PIPES, pH 7.0.

b

Data from ref. 57.

c

Data from ref. 60.

d

Data from ref. 56 

On the other hand, when the hydrophobic analogs were placed as a dangling base at the termini of the duplex, they actually stabilized the duplex more than their natural counterparts (Table 2). Similarly, hydrophobic pairs at the ends of a duplex increased the melting temperature more than a comparison A-T pair,57  and hydrophobic bases placed at the ends of a loop region in hairpin-loop DNA structures were shown to stabilize these structures.61 

Table 1.2

Melting temperatures for self-complementary DNA duplexes with a dangling natural base, nonpolar analog, or aromatic hydrocarbon.

Dangling residueaTm [°C]−ΔG°37 [kcal/mol]ΔΔG° stacking
Noneb 41.0 8.1±0.2  
Thymineb 48.1 9.2±0.2 1.1±0.2 
Adenineb 51.6 10.1±0.2 2.0±0.3 
Guaninec 51.5 9.4±0.2 1.3±0.2 
Cytosinec 46.2 9.1±0.2 1.0±0.2 
Fb 54.4 10.7±0.2 2.6±0.3 
Zb 54.6 11.1±0.2 3.0±0.3 
Hd 55.7 11.6±0.2 3.5±0.5 
Benzeneb 48.3 9.4±0.2 1.3±0.2 
Naphaleneb 56.2 10.9±0.2 2.8±0.3 
Phenanthreneb 57.3 10.7±0.2 2.6±0.3 
Pyreneb 64.1 11.4±0.2 3.3±0.3 
Dangling residueaTm [°C]−ΔG°37 [kcal/mol]ΔΔG° stacking
Noneb 41.0 8.1±0.2  
Thymineb 48.1 9.2±0.2 1.1±0.2 
Adenineb 51.6 10.1±0.2 2.0±0.3 
Guaninec 51.5 9.4±0.2 1.3±0.2 
Cytosinec 46.2 9.1±0.2 1.0±0.2 
Fb 54.4 10.7±0.2 2.6±0.3 
Zb 54.6 11.1±0.2 3.0±0.3 
Hd 55.7 11.6±0.2 3.5±0.5 
Benzeneb 48.3 9.4±0.2 1.3±0.2 
Naphaleneb 56.2 10.9±0.2 2.8±0.3 
Phenanthreneb 57.3 10.7±0.2 2.6±0.3 
Pyreneb 64.1 11.4±0.2 3.3±0.3 
a

Measured in the sequence d(XCGCGCG) in 1 M NaCl, 10 mM Na·phosphate, pH 7.0, 5 μM DNA.

b

Data from ref. 62.

c

Data from ref. 63.

d

Data from ref. 56 

The stability gained when hydrophobic bases were added to the ends of a duplex demonstrates that these bases are not lacking in stacking ability. However, it seems that the energy cost of pairing a natural base with a hydrophobic base arises from the energetic price of desolvation.57  When two complementary hydrophilic bases are paired in a duplex, solvation interactions between the polar groups and water are lost, but new hydrogen bonds are formed. When a hydrophilic base is paired with a hydrophobic base, the cost of the lost solvent interactions is not offset by the formation of new hydrogen bonds.

The smaller destabilization seen among the hydrophobic-hydrophobic base pairs could be explained through distortions to the normal B-form helix. While the Z-F pair was designed to conform to purine-pyrimidine geometry, Z is 0.5–1Å larger than A, so none of the hydrophobic base pairs perfectly match the geometry of their natural counterparts. Such an explanation is supported by the finding that multiple hydrophobic base pairs within a helix do not cause further destabilization,57  indicating that it is not the base pairs themselves that are destabilizing.

The ability of hydrophobic nucleobase mimics to stack relatively well in certain contexts in a DNA helix led to an effort to understand base stacking more fully by using a series of nonpolar aromatic hydrocarbons as bases.62  Benzene, naphthalene, phenanthrene, and pyrene nucleosides were incorporated as unpaired terminal residues in a self-complementary DNA sequence, and the stability of the resulting duplexes were compared (Table 2). It was found that in general, increasing the size of the aromatic residue led to improved helix stability. This could be due to both the increased ability for favorable stacking with neighboring base pairs and to avoidance of exposure of a larger hydrophobic surface area to the solvent.63 

The strong stacking ability of pyrene and its similarity in size to a natural base pair (220Å2vs. 269Å2 for an A-T pair, Fig. 2) led to the hypothesis that a pyrene nucleoside (P) might pair well internally with an abasic site (ϕ).64  Indeed, replacing an internal A-T pair with a P-ϕ pair led to only a slight destabilization, while pairing P or ϕ with a natural base were strongly destabilizing. This finding further suggests that hydrophobic base pairs are not inherently unstable; rather, destabilization results when a hydrophobic pair does not conform to the ideal geometry.

Figure 1.2

Structure of a pyrene-abasic site (P-ϕ) pair.

Figure 1.2

Structure of a pyrene-abasic site (P-ϕ) pair.

Close modal

Previous studies had shown that the hydrophobic base analogs discussed above could be incorporated by some polymerases with good efficiency and fidelity (see below), suggesting that size and shape can drive incorporation of a base partner. Incorporation of a pyrene nucleoside triphosphate at an abasic site would serve as a more extreme test of this steric exclusion model, pairing a very large base with the smallest partner possible. However, if successful, this pair could provide a useful means for the detection of abasic sites, which are a common form of DNA damage. dPTP was incorporated opposite an abasic site with more than one hundred times greater selectivity than the next best partner, dATP, and on the same order of magnitude as a natural base pair using the Klenow fragment of Escherichia coli DNA polymerase I (Kf exo-).65  These results clearly support the hypothesis that shape plays a critical role in the polymerase active site.

After incorporation of dPTP, the DNA polymerase stalled, resulting in strand termination; however, such stalling also occurred after incorporation of dATP as the abasic site partner. The polymerase stalling was used in Sanger sequencing of a synthetic DNA to successfully identify where abasic sites occurred.65 

The melting studies with hydrophobic base analogs described earlier in the chapter showed that hydrophobic bases paired best with one another (Table 1), suggesting that a more strongly hydrophobic base pair might increase the stability and selectivity of such a pair even further. Thus, the idea of a fluorous base pair was introduced, as fluorocarbons are generally more hydrophobic than their corresponding hydrocarbons.66  2,3,4,5-tetrafluorobenzene and 4,5,6,7-tetrafluoroindole were used as bases to create nucleoside analogs and incorporated into DNA oligonucleotides to test for their pairing ability, with benzene and indole base analogs used as controls (Fig. 3A).67  It was found that the fluorous base pairs did stabilize the helix more than either their analogous hydrocarbon pairs or mixed fluorocarbon-hydrocarbon pairs (Fig. 3B). The best pair, 4,5,6,7-tetrafluoroindole paired with itself, stabilized the helix by 3 kcal/mol relative to a T-C mismatch, about half of the stabilization provided by a T-A pair.67  These results support the hypothesis that solvophobic interactions serve as a driving force for nonpolar base pairs, allowing more of the hydrophobic surface to be buried away from water.

Figure 1.3

A) Molecular structure of hydrocarbon and fluorous nucleosides; B) relative stability of duplexes incorporating nonpolar bases. Adapted with permission from ref. 67. Copyright 2004 American Chemical Society.

Figure 1.3

A) Molecular structure of hydrocarbon and fluorous nucleosides; B) relative stability of duplexes incorporating nonpolar bases. Adapted with permission from ref. 67. Copyright 2004 American Chemical Society.

Close modal

Enzymatic DNA synthesis studies with the common bacterial DNA polymerase Kf exo- showed that the selectivity of fluorous pairs for one another was for the most part retained during polymerase extension of a template.68  The efficiency of insertion of 4,5,6,7-tetrafluoroindole nucleoside triphosphate opposite 2,3,4,5-tetrafluorobenzene in the template was within an order of magnitude of the insertion efficiency of a natural base pair (incorporation of A versus T). Additionally, fluorous pairing showed orthogonality to natural base pairing; these hydrophobic bases were incorporated more efficiently opposite one another than opposite natural bases, and likewise natural bases were not incorporated well as pairs for fluorous bases in the template. However, the 2,3,4,5-tetrafluorobenzene nucleoside triphosphate was not found to be selective in pairing with the fluorous indole over itself. Additionally, fluorous base pairs showed little or no extension by the polymerase, limiting the utility of these bases in their current form as an orthogonal base pair, at least with this native enzyme. Nevertheless, the successful design of a base pair of increased hydrophobicity confirmed the importance of stacking and desolvation effects in base pairing, and takes us a step further in our ability to engineer new base pairs.

While the pyrene-abasic site base pair was found to have similar stability to a natural base pair,64  the nonpolar nucleobase analog of T (F) paired with A was found to significantly destabilize the DNA duplex, and was not found to pair selectively with A over the other natural nucleobases (Table 1), consistent with the desolvation costs described above.57  It was therefore of interest how F and other nucleobase analogs would behave in incorporation studies with polymerases. While in the case of a P–ϕ pair, polymerases had been shown to incorporate an unnatural triphosphate with good efficiency and selectivity,65  this base pair had also shown good selectivity in duplex denaturation studies.64  With bases that matched in shape but caused destabilization of the helix and showed little selectivity in pairing with natural bases, how would the efficiency and selectivity of polymerases be affected? Use of nonpolar base analogs to probe polymerase activity has revealed insights into the importance of steric fit and minor groove contacts in this context.

The first studies were carried out with F in the template strand to look for selectivity in pairing with natural bases by Kf exo-. Strikingly, these results showed that dATP was incorporated with good efficiency and selectivity versus F (Fig. 4).34  The A-F pair was formed with only ∼5-fold lower efficiency than an A-T pair, and with similar selectivity. Furthermore, primers were also extended beyond this point without an observable pause after the A-F pair.34 

Figure 1.4

Efficiency of incorporation of unnatural and natural deoxynucleotides into DNA by E. coli pol I Kf exo-. A) Insertion efficiency with an unnatural templating base, with T for comparison; B) insertion efficiency of unnatural triphosphates, with dTTP for comparison. Data from refs 34, 71, and 74.

Figure 1.4

Efficiency of incorporation of unnatural and natural deoxynucleotides into DNA by E. coli pol I Kf exo-. A) Insertion efficiency with an unnatural templating base, with T for comparison; B) insertion efficiency of unnatural triphosphates, with dTTP for comparison. Data from refs 34, 71, and 74.

Close modal

Since the pairing of nucleotides opposite F is destabilizing regardless of the base, the polymerase results clearly showed that the enzyme enforces selectivity that the DNA alone does not. While this might support the hypothesis that active site steric effects play a crucial role in polymerase base-pairing, there are also other explanations that must be considered. The first is the long-known A-rule; A is incorporated more efficiently as the pair to an abasic lesion than the other natural bases.69,70  Thus, if F were simply being recognized as a “lesion”, it would not be surprising that A is inserted most efficiently opposite it. However, F codes for A 60 times better than an abasic site does.34  The clinching experiment involved turning the base pair around (inserting dFTP opposite the natural bases); the data showed that the F base is being read as an informational base rather than as a lesion. dFTP was incorporated with good efficiency versus A (only 40 times less efficient than the incorporation of dTTP vs. A) and with selectivity close to that of the T-A pair (Fig. 4).71,72 

Another explanation for the formation of the F-A pair could be that, although studies suggest F has minimal hydrogen-bonding ability,59,73  hydrogen bonds are still formed which make the pair a good match. The best way to test this was with the A analog, Z, as it has no polar functionality. With Z in the template, selective pairing with F and T both occurred (Fig. 4).74  The dZTP nucleotide paired efficiently only with F in the template.

While dZTP and dFTP are incorporated with efficiencies approaching those of natural bases, E. coli Kf exo- stalls after incorporation of Z.74  Notably, crystal structures and studies with polymerase mutants suggested that polymerases might form hydrogen bonds in the minor groove of the duplex just synthesized.75–79  The base Q was designed to serve as an analog to A with a hydrogen-bond acceptor in the minor groove (Fig. 5A).80  Incorporation and extension with Q or Z in the template and with dATP, dQTP, or dZTP showed that hydrogen bonding in the minor groove had negligible effect on incorporation efficiency, and minor groove hydrogen-bond contacts in the template strand did not affect extension of the duplex. However, significant stalling of the polymerase was found after incorporation of Z, while the nitrogen-containing Q was bypassed easily (Fig. 5B).80  Thus hydrogen-bond contacts in the minor groove play an important role in polymerase extension beyond a base pair.81–85 

Figure 1.5

A) Minor groove H-bond donor Q; B) autoradiogram showing efficient extension beyond A and Q but not Z. Adapted with permission from ref. 80. Copyright 1999 American Chemical Society.

Figure 1.5

A) Minor groove H-bond donor Q; B) autoradiogram showing efficient extension beyond A and Q but not Z. Adapted with permission from ref. 80. Copyright 1999 American Chemical Society.

Close modal

In addition to these and other studies carried out with enzymes in vitro, it was also of interest to test how analogs such as F and Q would be processed in the more complex environment of a cell. Bypass experiments were performed in Escherichia coli in which single-stranded phage plasmid with or without an analog (F or Q) was used to transfect the cells.29  The extent of bypass was measured by plaque formation, measured as a percentage of the plaques formed when a normal base was present. Under normal growth conditions, F was bypassed with about 30% efficiency, while Q showed a lower bypass efficiency of 6% (Fig. 6A). However, both bases showed remarkable fidelity – Q coded for T and F for A with similar levels to those with which the control G coded for C (Fig. 6B). When an SOS response was induced by irradiating cells with UV light, the fidelity of incorporation for all bases decreased, but the efficiency of bypass for Q, F, and a comparison abasic site were all significantly increased, suggesting lower selectivity among the enzymes involved in the SOS response.

Figure 1.6

Isosteres are bypassed in E. coli and code for the partner that is complementary in shape. A) Replication bypass efficiencies for templates containing F, Q, or an abasic site in comparison to G. B) Comparison of replication fidelity for G, F, and Q. Adapted from ref. 29. Copyright 2002 National Academy of Sciences, USA.

Figure 1.6

Isosteres are bypassed in E. coli and code for the partner that is complementary in shape. A) Replication bypass efficiencies for templates containing F, Q, or an abasic site in comparison to G. B) Comparison of replication fidelity for G, F, and Q. Adapted from ref. 29. Copyright 2002 National Academy of Sciences, USA.

Close modal

This experiment was significant from the synthetic biology perspective: it was the first demonstration of biological activity of unnatural bases in a living system. Overall, enzymatic and cellular studies with A and T analogs have served to underscore the critical role played by shape recognition in polymerase activity. These base mimics have served as tools to differentiate the roles of shape-matching and hydrogen-bonding in Kf exo- and other replicative DNA polymerases, in which they have been studied most extensively. However, ongoing work with additional enzymes is showing that different enzymes have varying tolerance for differences in shape and electrostatic interactions.86  Sensitivities to size and shape have been investigated using a series of finely tuned thymine analogs.

The importance of shape in replication leads to the hypothesis that enzymes of various types will display differences in their ability to tolerate bases of different sizes. For example, a low-fidelity enzyme may be predicted to have a larger or more flexible active site, providing a greater tolerance of base variation. In contrast, a high-fidelity enzyme would be expected to have a smaller, more rigid, active site. We created a “molecular ruler” to probe the tightness of enzyme fit by expanding the isosteres of thymine to range from toluene (H) to diiodotoluene (I) (Fig. 7).87,88  These isosteres vary in size in 0.2–0.4Å increments, but are designed to be as similar as possible in other properties.88 

Figure 1.7

Structure and bond lengths of thymidine analogs of increasing size. Data from ref. 89.

Figure 1.7

Structure and bond lengths of thymidine analogs of increasing size. Data from ref. 89.

Close modal

This set of thymidine mimics has proved useful in probing the steric sensitivity of active sites in a range of enzymes.89–96  A few examples can be used to illustrate the insights that can be provided. Kf exo- is a polymerase of relatively high fidelity, with an error rate of 10−3 to 10−4.97  When tested for incorporation with the unnatural analogs, dL, which is about 0.5Å larger than dT, was found to be the most efficient substrate and have the best fidelity for pairing with A (Fig. 8).89  However, both fidelity and efficiency of incorporation fell rapidly over an additional expansion of 0.35Å with dB and dI. The preference for dL suggests a larger-than-necessary active site, which would serve to provide some tolerance of mismatches, providing an evolutionary advantage. T7 DNA polymerase, like Kf exo-, is an A family polymerase, but in the presence of the E. coli protein thioredoxin shows much higher processivity and higher fidelity.98  T7 DNA polymerase in the presence of thioredoxin showed even higher sensitivity to slight steric changes than Kf exo-, and also a preference for slightly smaller shapes, pairing dFTP most efficiently as the incoming nucleotide.91  In contrast, Dpo4, a low-fidelity repair enzyme, showed markedly different behavior. Its preference, like Kf exo-, was for dL, but Dpo4 showed a wide tolerance for all analog sizes, with only a ∼35 fold difference in fidelity between dL and the worst-performing analog, dH.90  In addition, the fidelity of the nonpolar analogs for A was much lower than the fidelity for the A-T pair, suggesting that for this enzyme, additional factors such as electrostatic interactions are contributing to fidelity and incorporation efficiency.90 

Figure 1.8

Efficiency and fidelity of Kf exo- (diamond), T7 DNA pol (square), and Dpo4 (triangle) probed in sub-Angstrom increments with analogs of thymidine. A) and C) Incorporation of dATP with template X (X=H, F, L, B, or I). B) and D) Incorporation of dXTP with template A. Efficiency is calculated as ratio of A-T pair to best mismatch. Comparison A-T values are shown as open shapes at 1.2Å. Data from refs. 89, 90, and 91.

Figure 1.8

Efficiency and fidelity of Kf exo- (diamond), T7 DNA pol (square), and Dpo4 (triangle) probed in sub-Angstrom increments with analogs of thymidine. A) and C) Incorporation of dATP with template X (X=H, F, L, B, or I). B) and D) Incorporation of dXTP with template A. Efficiency is calculated as ratio of A-T pair to best mismatch. Comparison A-T values are shown as open shapes at 1.2Å. Data from refs. 89, 90, and 91.

Close modal

Importantly, the polymerase steric preferences from in vitro studies were confirmed in E. coli. Using a bypass assay, cells were found to tolerate dF and dL best, with about 50% bypass efficiency when compared with dT.89  All analogs were also primarily recognized as T. Interestingly, not only did nucleobase size play a role in polymerase bypass, but it also governed fidelity: the most efficient size (L) also displayed the highest fidelity in coding for A. This is consistent with the notion that a close steric fit in the active site yields most efficient and most selective DNA synthesis.

The analogs shown in Fig. 7 retain the shape of T but vary their size. Next we tested the effects of nucleobase shape, another important aspect of sterics. A series of mono- and dichloro-substituted bases having systematically varied shapes were developed and used in studies with E. coli Pol I Kf exo- (Fig. 9).99  Successful coding for A was primarily dependent on the presence of the 2-chloro substituent. Remarkably, the 3,4- and 2,3-dichloro analogs coded slightly more efficiently for T than for A, suggesting that substitution at the 3′ position eliminates specificity for A. The substitution of chlorine for hydrogen at the 3′ position corresponds to only a 0.7Å change, which suggests that the reduced activity of the analog Z relative to F may be in large part due to the relatively modest changes in structure made for this isostere.

Figure 1.9

A) Structure and bond lengths of chloro-substituted thymidine analogs. B) Efficiency of incorporation for dATP or dTTP (best mismatch) with monochloro template. C) Efficiency of incorporation for dATP or dTTP (best mismatch) with dichloro template. Reprinted with permission from ref. 99. Copyright 2006 WILEY-VCH Verlag GmbH & Co.

Figure 1.9

A) Structure and bond lengths of chloro-substituted thymidine analogs. B) Efficiency of incorporation for dATP or dTTP (best mismatch) with monochloro template. C) Efficiency of incorporation for dATP or dTTP (best mismatch) with dichloro template. Reprinted with permission from ref. 99. Copyright 2006 WILEY-VCH Verlag GmbH & Co.

Close modal

The ability to alter both the position and size of substituents on a DNA base mimic has proved remarkably effective for investigating sensitivity to shape and size within enzyme active sites. The results have added support to the steric exclusion model, suggesting a stringency for size and shape compatibility within the active sites of high-fidelity enzymes.

The work described above with nonpolar DNA analogs informed our understanding of how electrostatics, base shape, and size can affect enzyme incorporation and duplex stability. In that approach, small perturbations were made to natural DNA structure to evaluate biophysical and biochemical factors in DNA stability and replication. A second interest in our laboratory is the development of an unnatural genetic system based on, but alternative to, DNA. In this approach, we consider the properties that make DNA so successful as a genetic material, and try to replicate them in a synthetic system. In particular, the monomers must encode information, form stable primary and secondary structures, and support faithful replication of genetic material. Our synthetic system is based on the concept of expanded base pair size, and is called “expanded DNA” (xDNA). xDNA maintains DNA's sugar-phosphate backbone, but pairs the four natural DNA bases with a set of four size-expanded bases shown in Fig. 10, for a total of eight monomeric components.

Figure 1.10

Structures of expanded DNA nucleosides.

Figure 1.10

Structures of expanded DNA nucleosides.

Close modal

Because xDNA bases are similar in shape to natural bases, but are expanded by the width of a benzene ring (2.4Å), they can be useful in probing enzyme active site flexibility while conserving Watson-Crick hydrogen binding. Additionally, xDNA bases are inherently fluorescent, which could make them useful in nucleic acid probes or labels.

An expanded DNA nucleobase was first proposed and synthesized by Leonard and coworkers in the 1970s.100  They made ribonucleoside and ribonucleotide versions of a benzene-expanded adenosine,101,102  and went on to study their effects as mononucleotides with multiple enzymes.103,104  Expanded guanosine was also later synthesized.105  Our laboratory generalized this design to the pyrimidines as well, and conceived of the notion of combining the expanded bases with natural bases to make fully expanded helices. In this design, benzopurines would pair with pyrimidines, and benzopyrimidines with purines (Fig. 11A). This new genetic set has eight letters, and a sequence of mixed xDNA bases requires a complementary mixed sequence to match (Fig. 11B). On the other hand, a sequence consisting entirely of xDNA could complement a natural DNA sequence, forming an xDNA helix in the process.

Figure 1.11

A) An example of a benzopurine-pyrimidine pair (xA-T) and a purine-benzopyrimidine pair (G-xC). B) Complementary mixed strands can pair (left), and xDNA can pair with DNA (right), but a mixed xDNA/DNA strand cannot pair with DNA (center) and is thus orthogonal. Adapted with permission from ref. 122. Copyright 2007 American Chemical Society.

Figure 1.11

A) An example of a benzopurine-pyrimidine pair (xA-T) and a purine-benzopyrimidine pair (G-xC). B) Complementary mixed strands can pair (left), and xDNA can pair with DNA (right), but a mixed xDNA/DNA strand cannot pair with DNA (center) and is thus orthogonal. Adapted with permission from ref. 122. Copyright 2007 American Chemical Society.

Close modal

Synthesis of the benzopurines followed the route of Leonard106  up to intermediate 1 (Scheme 1). From there, preparation of dxA followed the route shown, with 4.3% overall percent yield in 8 steps.36  dxG proved more difficult, requiring radical deoxygenation of a ribose sugar after glycosylation rather than direct coupling to a deoxyribose sugar.107  It was prepared in 3.3% yield (14 steps). The benzopyrimidines were accessible via a Heck coupling to form the C–C glycosidic bond with the correct stereochemistry in good yield.36  dxT was achieved in 34% overall yield36  and served as a precursor for dxC, which required three additional steps.107 

Scheme 1.1

Synthesis of the expanded monomer dxA.a Reprinted with permission from ref. 36. Copyright 2004 American Chemical Society.

Scheme 1.1

Synthesis of the expanded monomer dxA.a Reprinted with permission from ref. 36. Copyright 2004 American Chemical Society.

Close modal

Both the deoxyribose and ribose108  versions of all expanded nucleosides have now been obtained in our laboratory, and the deoxyribonucleosides have also been converted to phosphoramidites for use in automated DNA synthesis and triphosphates for enzymatic studies described below.

Theoretical calculations predicted that xDNA bases would exist stably in the desired tautomeric conformation,36  with minor deviations from planarity in the bases (also found in normal DNA).109  Once synthesized, the expanded DNA bases were incorporated into both natural and expanded DNA duplexes to study properties such as pairing and stacking.

The incorporation of a single xDNA base in the center of a DNA duplex proved to be destabilizing, causing a loss of free energy of 0.3–1.7 kcal/mol (Table 3), as one might expect with a large change in size at the DNA/xDNA junctions.107,110  However, in most cases the bases retained selectivity for their Watson-Crick hydrogen-bonding partner by 1–4 kcal/mol. These data suggest that although the presence of an expanded base causes some unfavorable distortion to the helix, a face-to-face hydrogen-bonding conformation is adopted by the correct pair, rather than other possible conformations such as base-on-base intercalative stacking or a flipped-out base. CD spectra also confirm that B-form helical structure is retained.110  Multiple substitutions within the center of the duplex do not cause additional destabilization (Table 3),110  indicating that the distortion of the backbone necessary to accommodate the larger base-pair size is what causes the change in free energy, rather than the expanded geometry itself.

Table 1.3

Melting temperatures for DNA duplexes containing xDNA bases.

Base Pair X-YaTm [°C]ΔTm [°C]−ΔG°25 [kcal/mol]
T-Ab 40.7 – 9.3±0.1 
A-T 40.4 – 9.2±0.1 
T-xA 35.8 4.9 8.1±0.1 
G-xA 29.3 11.4 6.6±0.1 
C-xA 29.9 10.8 6.8±0.2 
A-xA 27.8 12.9 6.4±0.1 
abasic-xA 29.6 11.1 6.8±0.2 
(T-xA)2 35.5 5.2 8.1±0.1 
(T-xA)3 37.2 3.5 8.4±0.1 
A-xT 35.2 5.2 8.0±0.2 
T-xT 31.3 9.1 7.0±0.1 
G-xT 28.2 12.2 6.2±0.2 
C-xT 25.5 14.9 5.6±0.2 
abasic-xT 21.1 19.3 5.1±0.2 
C-Gc 43.1 – 9.7±0.8 
G-C 45.6 – 10.4±0.8 
G-xC 41.2 4.4 9.4±0.2 
A-xC 25.0 20.6 5.9±0.4 
C-xC 29.9 15.7 6.6±0.2 
T-xC 28.5 17.1 6.7±0.2 
abasic-xC 26.5 19.1 6.0±0.4 
C-xG 36.0 7.1 8.2±0.8 
A-xG 25.9 17.2 6.1±0.6 
G-xG 28.7 14.4 7.0±0.7 
T-xG 28.8 14.3 6.3±0.6 
abasic-xG 28.3 14.8 6.4±0.6 
Base Pair X-YaTm [°C]ΔTm [°C]−ΔG°25 [kcal/mol]
T-Ab 40.7 – 9.3±0.1 
A-T 40.4 – 9.2±0.1 
T-xA 35.8 4.9 8.1±0.1 
G-xA 29.3 11.4 6.6±0.1 
C-xA 29.9 10.8 6.8±0.2 
A-xA 27.8 12.9 6.4±0.1 
abasic-xA 29.6 11.1 6.8±0.2 
(T-xA)2 35.5 5.2 8.1±0.1 
(T-xA)3 37.2 3.5 8.4±0.1 
A-xT 35.2 5.2 8.0±0.2 
T-xT 31.3 9.1 7.0±0.1 
G-xT 28.2 12.2 6.2±0.2 
C-xT 25.5 14.9 5.6±0.2 
abasic-xT 21.1 19.3 5.1±0.2 
C-Gc 43.1 – 9.7±0.8 
G-C 45.6 – 10.4±0.8 
G-xC 41.2 4.4 9.4±0.2 
A-xC 25.0 20.6 5.9±0.4 
C-xC 29.9 15.7 6.6±0.2 
T-xC 28.5 17.1 6.7±0.2 
abasic-xC 26.5 19.1 6.0±0.4 
C-xG 36.0 7.1 8.2±0.8 
A-xG 25.9 17.2 6.1±0.6 
G-xG 28.7 14.4 7.0±0.7 
T-xG 28.8 14.3 6.3±0.6 
abasic-xG 28.3 14.8 6.4±0.6 
a

Measured in the sequence d(CTTTTCXTTCTT)·d(AAGAAYGAAAAG) in 100 mM NaCl, 10 mM MgCl2, 10 mM Na·PIPES, pH 7.0.

b

Data from ref. 110.

c

Data from ref. 107 

xDNA duplexes showed two-state cooperative melting and generally higher melting temperatures than their natural DNA counterparts (Fig. 12).111,112  This may be explained by the enhanced stacking ability of xDNA bases, which was measured to be energetically more than twice that of the corresponding natural bases.107,110  Additionally, high selectivity, strikingly similar in magnitude to that of natural DNA, was seen in xDNA against single mismatches (Fig. 13).112  Such selectivity is a necessary trait for a replicable genetic system, and will also be useful in xDNA probes designed to complement natural DNA or RNA sequences.

Figure 1.12

Thermal denaturation plot showing increased melting temperature of xDNA duplex with sequence d(xAxTCAxCTxGxTGCp)·d(xGxCACxAGxTxGATp). Solid line shows melt curve for xDNA; dashed line is analogous DNA melt. Reprinted with permission from ref. 112. Copyright 2005 WILEY-VCH Verlag GmbH & Co.

Figure 1.12

Thermal denaturation plot showing increased melting temperature of xDNA duplex with sequence d(xAxTCAxCTxGxTGCp)·d(xGxCACxAGxTxGATp). Solid line shows melt curve for xDNA; dashed line is analogous DNA melt. Reprinted with permission from ref. 112. Copyright 2005 WILEY-VCH Verlag GmbH & Co.

Close modal
Figure 1.13

Sequence selectivity of A) xDNA compared to B) DNA, based on differences in temperatures of single-mismatch strands. Reprinted from ref. 112. Copyright 2005 WILEY-VCH Verlag GmbH & Co.

Figure 1.13

Sequence selectivity of A) xDNA compared to B) DNA, based on differences in temperatures of single-mismatch strands. Reprinted from ref. 112. Copyright 2005 WILEY-VCH Verlag GmbH & Co.

Close modal

While preliminary studies showed cooperative melting behavior,111  it was of interest to investigate the structure of xDNA more thoroughly. Duplex formation was predicted due to the retained stacking and hydrogen-bonding abilities, but it was unclear how the change in size might affect backbone conformation and alter structural preferences. NMR studies with a self-complementary sequence containing all xA-T pairs113  and a more complex sequence with all eight nucleotides114  showed that both formed right-handed antiparallel helices in solution (Fig. 14). Likewise, both matched DNA duplexes in having anti glycosidic bond conformations and 2′-endo sugar conformations, but displayed wider major and minor grooves than DNA. However, some differences between the two xDNA duplexes were also observed. The xA-T duplex showed a slightly smaller rise per base pair than natural DNA (3.1Å vs. 3.4Å) and steeper helix pitch (37° vs. 34°), along with deeper major and minor grooves.113  In contrast, the second duplex (containing all eight bases) showed a larger rise per base pair (4.0Å vs. 3.4Å) and shallower pitch (30° vs. 34°), along with shallower major and minor grooves and a more dynamic structure.114  Within such short (10 bp) duplexes of different composition, such variation is not surprising. The geometric differences among the base pairs likely account for the differences in the major and minor groove depth.114  Additionally, the reduced pKa of the imino proton of dxG relative to dG may have contributed to the structural movement of the more complex sequence, as it appeared that partial deprotonation could occur at the pH at which the experiments were performed.114 

Figure 1.14

Space-filling structure of xDNA (left) in comparison to DNA (right). A view of the major groove view on top; the minor groove is shown on the bottom. Adapted with permission from ref. 113. Copyright 2004 American Chemical Society.

Figure 1.14

Space-filling structure of xDNA (left) in comparison to DNA (right). A view of the major groove view on top; the minor groove is shown on the bottom. Adapted with permission from ref. 113. Copyright 2004 American Chemical Society.

Close modal

Additional structural studies were carried out in solution using UV and fluorescence measurements.112,115  Multiple complementary sequences were investigated, showing cooperative melting and 1:1 stoichiometry in most cases, although there was also evidence for triplex formation between a poly(A) sequence and a poly(xT) sequence,115  which has analogous examples in DNA.116  Interestingly, ionic strength dependence was found to be similar between DNA and xDNA, despite the greater distance between phosphate backbones in xDNA.115 

Research into the structural properties of xDNA shows that it retains two of the key characteristics that make DNA so successful as a genetic material: a stable helical secondary structure, and selective pairing properties. This characterization has made it clear that the DNA backbone structure is adaptable to significant changes in base pair geometry. However, it remained to be seen whether the expanded information-encoding capabilities of xDNA could be harnessed. To that end, polymerase and cellular studies have provided some early insight into whether xDNA might be tolerated for replication.

As described in the earlier part of this chapter, previous studies had shown that some polymerases have an acute sensitivity to size in the fidelity and efficiency of incorporation;89,91,99  natural polymerases are highly evolved to function with natural DNA, and so it was unclear whether they would be able to tolerate the larger xDNA nucleotides. Nevertheless, it was important to establish if some polymerase activity was present, which would improve prospects for modifying that activity to better accommodate xDNA and would also give further insight into the flexibility and size of enzyme active sites.

Initial tests involved two enzymes: Kf exo-, a relatively high-fidelity enzyme, and Dpo4, a repair enzyme responsible for the extension of mismatches and lesions. In all cases, Kf showed some preference for insertion of the correct base-pairing partner across from an xDNA base in the template strand; Dpo4 showed a modest selectivity for the correct base pair in all cases except for xT, which was paired equally well with A and with T.117  However, incorporation efficiencies were at least two orders of magnitude reduced from that of natural base pairs, and fidelity was also generally much lower, with less than a ten-fold preference for the correct pair over a mismatch observed in several cases. The ability of the enzymes to extend a DNA-xDNA pair was also considered; while Kf showed very little extension ability, Dpo4 was successful in extending DNA-xDNA pairs, and in most cases showed selectivity, extending a correctly-matched base-pair better than a mismatched one.117  Additionally, Dpo4 showed the ability to insert the correct base to extend a DNA primer on an all-xDNA template, suggesting that it is flexible enough to accommodate the larger xDNA helix to some degree.

Studies were also carried out in E. coli to determine whether the replicability of single or few xDNA substitutions in a DNA strand might fare better or worse in the presence of complex cellular machinery. An initial study, in which the ability of E. coli to bypass an expanded base to replicate a single-stranded phage genome was measured, found that xA and xT were efficiently bypassed under normal cellular conditions (74% and 80%, respectively), while xC (29%) and xG (11%) performed somewhat more poorly (Fig. 15).30  xA and xC were also recognized as their DNA base counterparts, while xG and xT were found to be read primarily as A. The prevalence of the xT-T mismatch here and with Dpo4 is attributed to the ability of these two bases to form a hydrogen-bonded structure closer in width to the natural DNA helix than an expanded base pair.

Figure 1.15

E. coli are capable of bypassing and reading expanded nucleobases. A) Replication bypass efficiencies for templates containing expanded bases in comparison to G; B) comparison of replication fidelity for G and expanded bases; C) proposed structure for xT-T mispair. Adapted from ref. 30. Copyright 2009 WILEY-VCH Verlag GmbH & Co.

Figure 1.15

E. coli are capable of bypassing and reading expanded nucleobases. A) Replication bypass efficiencies for templates containing expanded bases in comparison to G; B) comparison of replication fidelity for G and expanded bases; C) proposed structure for xT-T mispair. Adapted from ref. 30. Copyright 2009 WILEY-VCH Verlag GmbH & Co.

Close modal

The capacity of enzymes to successfully bypass xDNA at all was considered encouraging, and prompted investigation as to whether xDNA bases could be read successfully to encode for amino acids in a protein. xDNA bases were incorporated into both strands of a plasmid coding for green fluorescent protein (GFP) and transfected into E. coli.31  Although colony yields were lower when plasmids contained xDNA, green colonies were obtained for all substitutions investigated, including up to three expanded nucleotides per strand. In addition, sequencing of plasmid copies showed that in all cases, the xDNA bases were recognized as encoding for their appropriate partner. Knockout strains of E. coli indicated that repair enzymes did not appear to be necessary in the processing of xDNA nucleotides; while Y family polymerases showed some effect, the results suggested the involvement of a mixture of enzymes in processing the xDNA bases.

Perhaps the greatest significance of the results was the finding that a non-Watson-Crick genetic set could encode amino acids of a protein in a living cell. This was the first example of such an achievement, and bodes well for future studies in synthetic biology.

While some successes have been achieved in replicating isolated xDNA bases or small segments of xDNA, we expect that much more work will be needed to efficiently replicate xDNA in vitro and in vivo, and to determine whether reliable fidelity can be achieved. We envision that both screening a range of polymerases for the best activity with xDNA and polymerase evolution techniques will be required to be successful in this regard.

In addition to their ability to form highly stable and selective helices, the expanded bases’ inherent fluorescence gives them a unique advantage over most DNA analogs as tools for detection and labeling. xDNA bases are blue fluorophores, with emission maxima around 380–410nm and high quantum yields of 0.30–0.52.36,107  While other fluorescent DNA base analogs are known,118  xDNA may present unique properties through its ability to base pair with natural DNA and its strong stacking propensity.

Studies of homooligomers of 1–4 expanded bases at the 5′ end of a DNA oligomer led to the discovery of some interesting properties.119  While xT and xG showed self-quenching behavior when multiple monomers were incorporated, two or more adjacent xA nucleotides led to a decrease in the monomer emission peak of 390nm and the appearance of a new peak at 520nm, consistent with excimer formation (Fig. 16). xC also showed a surprising result, with increased emission relative to the monomer when more than two xC nucleotides were present. Additionally, hybridizing the xDNA-containing strands to a complement that paired natural bases with one or more of the expanded bases showed that some of the properties were changed upon hybridization. For example, xC and xT were quenched when incorporated opposite G; xG showed enhanced fluorescence opposite A. Studies of these simple systems suggest that quite complicated behavior could arise in more complex sequences; however, xDNA clearly has responsive properties that could be useful in specific sequence detection.

Figure 1.16

Emission spectra of 10mer DNAs terminating in 1-4 xN nucleotides. A) Emission of (xA)n strands (ex. 333nm); B) emission of (xG)n strands (ex. 320nm); C) emission of (xT)n strands (ex. 321nm); D) emission of (xC)n strands (ex. 330nm). Reprinted with permission from ref. 119. Copyright 2008 American Chemical Society.

Figure 1.16

Emission spectra of 10mer DNAs terminating in 1-4 xN nucleotides. A) Emission of (xA)n strands (ex. 333nm); B) emission of (xG)n strands (ex. 320nm); C) emission of (xT)n strands (ex. 321nm); D) emission of (xC)n strands (ex. 330nm). Reprinted with permission from ref. 119. Copyright 2008 American Chemical Society.

Close modal

While the previous work incorporated expanded nucleotides by DNA synthesizer at the terminus of a DNA strand, we wondered whether an enzyme might do the job instead. Terminal deoxytransferase (TdT) is an enzyme that extends DNA primers by incorporating nucleoside triphosphates in the absence of a template, and is used in assays to label DNA fragments. We found that TdT showed incorporation efficiencies of xDNA triphosphates comparable to those of natural DNA triphosphates, with 3–15 incorporations of expanded bases seen in most cases.120  Additionally, enzymatic reactions in solution or on beads showed the expected increase in fluorescence with incorporation of xC and the emergence of longer-wavelength emission with xA in the oligomeric products.

When pondering the origins of life on earth, one wonders why nature chose DNA as the genetic material. Are there other options that could be viable? In order to explore this question, a relatively minor modification, namely the addition of a benzene ring to each base pair, was adopted for the creation of a new genetic set – xDNA. While work on successful replication of xDNA is ongoing, we have verified that this new genetic material shares some of the key features of DNA – namely, a stable, antiparallel helical secondary structure and discrimination between pairing partners necessary for the faithful encoding of information. In addition, preliminary results toward replication are promising; in E. coli, these bases can faithfully direct enzymatic incorporation of the complementary base partner, and ultimately encode amino acids of a functional protein.

xDNA's inherent fluorescence, emergent fluorescent properties in oligomers, and strong pairing ability with DNA make it promising as a tool; for example, xDNA tags could be used in affinity purification, or fluorescent sequence-specific tags could be developed.

The main hindrance in both the development of polymerases for xDNA replication and the creation of tools and probes using xDNA is the difficulty of synthesis, particularly of the expanded purine nucleotides. Thus new synthetic routes are worthy of future exploration.

Studies of xDNA and nonpolar nucleobase isosteres have provided useful insights into basic biochemical questions, and are pointing the way to practical uses. Our steric studies of high-fidelity polymerases have shown that tight active sites regulate fidelity and efficiency even in the absence of hydrogen bonds, and this has led to the development of PCR-amplifiable, specific non-hydrogen bonding base pairs, as exemplified elegantly by the laboratories of Romesburg and Hirao.121  Similar steric studies with low-fidelity repair enzymes showed the importance of hydrogen bonds for function in that class of polymerases, and this subsequently led to the finding of assisted replication of xDNA by such flexible enzymes, not only in vitro but in living cells as well.

Overall, our continuing work in the field of chemical synthetic biology leads us to a better understanding of the natural genetic system, and many of the components required for its successful operation. As we tease apart these factors, we come closer to the development of a new genetic set upon which, one day, new living systems can be based.

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