Aaron M. Leconte

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogues, we performed a detailed structure-activity relationship analysis, which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.

Discovery and biological characterization of geranylated RNA in bacteria.

A general mass spectrometry-based screen for unusually hydrophobic cellular small-molecule RNA conjugates revealed geranylated RNA in E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella thyphimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNAGluUUC, tRNALysUUU, tRNAGlnUUG at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation levels can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNAGluUUC, tRNALysUUU, and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution.

To what extent are evolutionary outcomes determined by a populations recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories (“pathways”) demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.

Optimization of the Pyridyl Nucleobase Scaffold for Polymerase Recognition and Unnatural Base Pair Replication

As part of an effort to increase both the biological and biotechnological applications of DNA, we[1–5] and others[6–9] have explored the DNA polymerase-mediated replication of a wide range of unnatural base pairs. In our initial efforts we examined large, aromatic, unnatural nucleotides, both as self pairs of two identical nucleotides and heteropairs of different nucleotides.[1–5,10,11] While several of these unnatural base pairs are efficiently synthesized (i.e. by insertion of the unnatural dNTP opposite its partner in the template) by the exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (Kf), none are efficiently extended (i.e. by continued primer elongation), most likely due to interstrand nucleobase intercalation and distortion of the primer terminus.[10] Thus, a range of nucleotides bearing smaller phenyl-based nucleobases that should be incapable of intercalation were explored, and several modifications that facilitate extension were identified.[1–4] Of these, aza-substitution at the 2 position (2Py, Figure 1) appears to be the only modification that facilitates self pair extension without significantly facilitating mispairing.[3]

Directed Evolution of DNA Polymerases for Next‐Generation Sequencing

We present the application of an activity-based phage display method to identify DNA polymerases tailored for next generation sequencing applications. Using this approach, we identify a mutant of Taq DNA polymerase that incorporates the fluorophore-labeled dA, dT, dC, and dG substrates ~50 to 400-fold more efficiently into scarred primers in solution and that also demonstrates significantly improved performance under actual sequencing conditions.

Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase

Trypanothione reductase (TR) is found in the trypanosomatid parasites, where it catalyses the NADPH-dependent reduction of the glutathione analogue, trypanothione, and is a key player in the parasite’s defenses against oxidative stress. TR is a promising target for the development of antitrypanosomal drugs; here, we report our synthesis and evaluation of compounds 3–5 as low micromolar Trypanosoma cruzi TR inhibitors. Although 4 and 5 were designed as potential irreversible inhibitors, these compounds, as well as 3, displayed reversible competitive inhibition. Compound 3 proved to be the most potent inhibitor, with a Ki = 2 µM.

Polymerase recognition and stability of fluoro-substituted pyridone nucleobase analogues.

Recently much effort has been focused on designing unnatural base pairs that are stable and replicated by DNA polymerases with high efficiency and fidelity. This work has helped to identify a variety of nucleobase properties that are capable of mediating the required interbase interactions in the absence of Watson–Crick hydrogen‐bonding complementarity. These properties include shape complementarity, the presence of a suitably positioned hydrogen‐bond donor in the developing minor groove, and fluorine substitution. In order to help characterize how each factor contributes to base pairing stability and replication, we synthesized and characterized three fluoro‐substituted pyridone nucleoside analogues, 3 FP, 4 FP, and 5 FP. Generally, we found that the specific fluorine substitution pattern of the analogues had little impact on unnatural pair or mispair stability, with the exception of mispairs with dG, which were also the most stable. The mispair between dG and 3 FP was less stable than that with 4 FP or 5 FP, which likely resulted from specific interbase interactions. While fluorine substitution had little impact on the synthesis of the unnatural base pairs, it significantly enhanced mispairing with dG. Remarkably, the mispair between dG and 3 FP was the most efficiently synthesized, due to a favorable entropy of activation, which possibly resulted from the displacement of water molecules from dG in the phosphoryl transfer transition state. The more efficient synthesis of the 3 FP–dG mispair, despite its being the least stable of the three, suggests that the determinants of synthesis and stability are distinct. Finally, we found that fluorine substitution significantly increased the rate at which the pyridone‐based unnatural base pairs were extended; this suggests that both minor groove hydrogen‐bond acceptors and fluorine substituents could be used to simultaneously optimize unnatural base pairs.

Amplify this! DNA and RNA get a third base pair

DNA containing a new unnatural base pair may be amplified by PCR and transcribed into RNA, potentially increasing the diversity available from nucleic acids.

Chemical biology: A broader take on DNA

Slipping in extra benzene rings creates a broader DNA double helix that is similar to, but different from, natural DNA. Importantly, it can encode more genetic information — and that could have wide implications.

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more‐diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2′‐modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2′F‐, 2′OH‐, 2′OMe‐, and 3′OMe‐modified DNA as well as for reverse transcription of 2′OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2′OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.