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 being an essential first step towards 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 analogs, 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.
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.
To what extent are evolutionary outcomes determined by a population's 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. directed evolution | evolutionary biology | tape of life
In the groove: In an effort to develop unnatural base pairs, pyridyl nucleoside analogues were synthesized and characterized (see structures). An α‐glycosidic nitrogen atom provides an H‐bond acceptor that does not significantly facilitate pairing with natural nucleobases. However, it forms minor‐groove H‐bonds with water molecules and DNA polymerases that optimize the stability and replication, respectively, of the unnatural base pair.
A non-natural base pair, which is stable in duplex DNA and enzymatically synthesized with high efficiency and selectivity, would greatly expand the utility of nucleic acids, both in terms of their genetic-coding capacity and chemical functionality. Unlike the natural base pairs, a third base pair does not necessarily require H-bonding,[1] and we, [2] and others, [3] have demonstrated that hydrophobic and van der Waals forces can mediate the selective interbase interactions required for basepair stability and synthesis.Our early efforts focused on nucleotides bearing nucleobase analogues with large extended aromatic surfaces. A variety of self-pairs (formed between the same analogue) and heteropairs (formed between different analogues) were identified. These pairs were both stable in duplex DNA and efficiently synthesized by the exonuclease deficient Klenow fragment of DNA polymerase I (Kf).[2a, 4] However, replication was consistently limited by poor extension (i.e. continued primer elongation after synthesis of the non-natural pair), which we reasoned could be the result of interbase intercalation of the large aromatic scaffolds that distort the newly formed primer terminus and prevent its efficient extension. Our second-generation non-natural base pairs were formed between nucleotides that bear small, aromatic nucleobase analogues that were designed to be too small to intercalate and therefore more likely to interact edge on. Although several base pairs have been found that are more efficiently extended by Kf, the rates remain insufficient. [2b, 5] Further-
Protein evolution is a critical component of organismal evolution and a valuable method for the generation of useful molecules in the laboratory. Few studies, however, have experimentally characterized how fundamental parameters influence protein evolution outcomes over long evolutionary trajectories or multiple replicates. In this work, we applied phage-assisted continuous evolution (PACE) as an experimental platform to study evolving protein populations over hundreds of rounds of evolution. We varied evolutionary conditions as T7 RNA polymerase evolved to recognize the T3 promoter DNA sequence and characterized how specific combinations of both mutation rate and selection stringency reproducibly result in different evolutionary outcomes. We observed significant and dramatic increases in the activity of the evolved RNA polymerase variants on the desired target promoter after 96 hours of selection, confirming positive selection occurred under all conditions. We used high-throughput sequencing to quantitatively define convergent genetic solutions, including mutational “signatures” and non-signature mutations that map to specific regions of protein sequence. These findings illuminate key determinants of evolutionary outcomes, inform the design of future protein evolution experiments, and demonstrate the value of PACE as a method to study protein evolution.
Genetic information is encoded by, but potentially not limited to a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogs that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogs and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containg the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations are consistent with an increased affinity for the DNA primer-template containing the selfpair.The biological system of information storage, based on the selective Watson-Crick hydrogen bonding (H-bonding) of adenine with thymine (dA:dT base pair) and guanine with cytosine (dG:dC base pair), has been conserved throughout nature. Expansion of the alphabet to contain a third base pair would allow additional information to be encoded in DNA and would also enable a variety of in vitro experiments using unnatural nucleic acids. 1 A priori, there is no reason to assume that the requirements for duplex stability and replication must limit the genetic To whom correspondence should be addressed. E-mail floyd@scripps.edu. One strategy for improving the replication of these unnatural base pairs is based on their further derivatization with heteroatoms or alkyl substituents. 6, 7 This strategy mimics the optimization of the natural bases in nature. However, in nature, base pair optimization proceeded simultaneously with DNA polymerase evolution. Thus, any expansion of the genetic code is expected to be facilitated by optimizing both the unnatural nucleobase analogs and the polymerases that replicate them. Here, we report our initial efforts towards the directed evolution of polymerases that more efficiently synthesize DNA containing the PICS self-pair. NIH Public AccessPreviously, we reported a phage display selection system designed to evolve DNA or RNA polymerases with novel activities. 8,9 We used the selection system to evolve variants of Sf that efficiently synthesize RNA 8 or DNA containing C2'-O-methyl modified nucleotides. 9The selection system is ba...
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