Expanding the substrate repertoire of a DNA polymerase by directed evolution

Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.     

Solution Structure,Mechanism of Replication,and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge‐on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue d MMO2 , which forms an unnatural base pair with the nucleotide analogue d 5SICS . Replacing the para‐methyl substituent of d MMO2 with an annulated furan ring (yielding d FMO ) has a dramatically negative effect on replication, while replacing it with a methoxy (d DMO ) or with a thiomethyl group (d TMO ) improves replication in both steady‐state assays and during PCR amplification. Thus, d TMO –d 5SICS , and especially d DMO –d 5SICS , represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental d MMO2 –d 5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.     

Redox-coupled dynamics and folding in cytochrome c

Cytochrome c functions as an electron carrier in the mitochondrial electron-transport chain using the Fe(II)-Fe(III) redox couple of a covalently attached heme prosthetic group, and it has served as a paradigm for both biological redox activity and protein folding. On the basis of a wide variety of biophysical techniques, it has been suggested that the protein is more flexible in the oxidized state than in the reduced state, which has led to speculation that it is the dynamics of the protein that has been evolved to control the cofactor's redox properties. To test this hypothesis, we incorporated carbon-deuterium bonds throughout cytochrome c and characterized their absorption frequencies and line widths using IR spectroscopy. The absorption frequencies of several residues on the proximal side of the heme show redox-dependent changes, but none show changes in line width, implying that the flexibility of the oxidized and reduced proteins is not different. However, the spectra demonstrate that folded protein is in equilibrium with a surprisingly large amount of locally unfolded protein, which increases with oxidation for residues localized to the proximal side of the heme. The data suggest that while the oxidized protein is not more flexible than the reduced protein, it is more locally unfolded. Local unfolding of cytochrome c might be one mechanism whereby the protein evolved to control electron transfer.     

Site‐Specifically Arraying Small Molecules or Proteins on DNA Using An Expanded Genetic Alphabet

A class of replicable unnatural DNA base pairs formed between d 5SICS and either d MMO2 , d DMO , or d NaM were developed. To explore the use of these pairs to produce site‐specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase‐mediated replication, and subsequent site‐specific modification of the amplified DNA by Click chemistry is reported. With the d 5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the d MMO2 and d DMO analogues, the d MMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether‐based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site‐selectively attach a biotin tag to the amplified DNA. Finally, we use d 5SICS^CO –d NaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials.     

Minor groove hydrogen bonds and the replication of unnatural base pairs

As part of an effort to expand the genetic alphabet, we examined the synthesis of DNA with six different unnatural nucleotides bearing methoxy-derivatized nucleobase analogues. Different nucleobase substitution patterns were used to systematically alter the nucleobase electronics, sterics, and hydrogen-bonding potential. We determined the ability of the Klenow fragment of E. coli DNA polymerase I to synthesize and extend the different unnatural base pairs and mispairs under steady-state conditions. Unlike other hydrogen-bond acceptors examined in the past, the methoxy groups do not facilitate mispairing, implying that they are not recognized by any of the hydrogen-bond donors of the natural nucleobases; however, they do facilitate replication. The more efficient replication results largely from an increase in the rate of extension of primers terminating at the unnatural base pair and, interestingly, requires that the methoxy group be at the ortho position where it is positioned in the developing minor groove and can form a functionally important hydrogen bond with the polymerase. Thus, ortho methoxy groups should be generally useful for the effort to expand the genetic alphabet.     

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

A variety of IR‐active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon‐deuterium bonds (C?D), cyano groups (CN), and azides (N₃); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N‐terminal Src homology 3 domain of the murine adapter protein Crk‐II (nSH3) with C?D bonds site‐selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two‐state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N₃ probes at five of the same positions. In agreement with previous studies, the position‐dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface‐exposed positions. Thus, unlike C?D labels, which are both sensitive to their environment and non‐perturbative, CN and N₃ probes should be used with caution.     

Synthesis and characterization of the arylomycin lipoglycopeptide antibiotics and the crystallographic analysis of their complex with signal peptidase

Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.     

Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues

In an effort to develop an orthogonal third base pair for the storage of genetic information, thiophene and furan heterocycles have been examined as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases. These results expand the potential nucleobase analogues with which the genetic alphabet may be expanded to include five-membered-ring heterocycles.