Gil Tae Hwang

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.

Optimization of an Unnatural Base Pair toward Natural-Like Replication

Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivatives are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM:d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the minimum overall fidelity ranges from 10(3) to 10(4). In addition, the data allow us to propose a generalized model of unnatural base pair replication, which should aid in further optimization of the unnatural base pair and possibly in the design of additional unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.

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 dMMO2, which forms an unnatural base pair with the nucleotide analogue d5SICS. Replacing the para-methyl substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically negative effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO-d5SICS, and especially dDMO-d5SICS, 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 dMMO2-d5SICS 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.

Substituent effects on the pairing and polymerase recognition of simple unnatural base pairs

As part of an effort to develop stable and replicable unnatural base pairs, we have evaluated a large number of unnatural nucleotides with predominantly hydrophobic nucleobases. Despite its limited aromatic surface area, a nucleobase analog scaffold that has emerged as being especially promising is the simple phenyl ring. Modifications of this scaffold with methyl and fluoro groups have been shown to impact base pair stability and polymerase recognition, suggesting that nucleobase shape, hydrophobicity and electrostatics are important. To further explore the impact of heteroatom substitution within this nucleobase scaffold, we report the synthesis, stability and polymerase recognition of nucleoside analogs bearing single bromo- or cyano-derivatized phenyl rings. Both modifications are found to generally stabilize base pair formation to a greater extent than methyl or fluoro substitution. Moreover, polymerase recognition of the unnatural base pairs is found to be very sensitive to both the position and nature of the heteroatom substituent. The results help identify the determinants of base pair stability and efficient replication and should contribute to the effort to develop stable and replicable unnatural base pairs.

Synthesis and characterization of new polyimides containing calix[4]arenes in the polymer backbone

Two diaminocalix[4]arene monomers were synthesized from p-tert-butylcalix[4]arene through a 4-step reaction sequence. New copoly(amic acid)s containing calix[4]arene moieties on the polymer backbone were successfully synthesized in N-methyl-2-pyrrolidone by polycondensations of 4,4′-oxydiphthalic anhydride (ODPA) with the diaminocalix[4]arene monomers using 4,4′-oxydiphenylene diamine (ODA) as a comonomer. These copoly(amic acid)s were soluble in aprotic polar solvents, so that they can be processed in various ways. The copoly(amic acid) precursors were thermally converted to the corresponding copolyimides in films. The copolyimide films are amorphous, but insoluble in common solvents. They are thermally stable up to 366°C. The copolyimides exhibit relatively high TECs, low Tgs, low refractive index, low dielectric constant, low optical anisotropy, low dielectric anisotropy, and low water uptake, compared to those of conventional ODPA-ODA polyimide. These property characteristics were interpreted in regard to bulky, cone-like calix[4]arene moieties and their effects on the chain conformation and morphological structure. The processability and property characteristics support that both of the copolyimides containing calix[4]arene moieties are potential candidate materials suitable for membranes, antioxidant additives, chemical sensor devices, and microelectronic devices.

Unnatural substrate repertoire of A, B, and X family DNA polymerases.

As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examined the ability of five different polymerases to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five additional polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9(o)N-7, and the family X polymerase, human polymerase beta. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examined, suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is especially noteworthy due to strong mispair discrimination.

Synthesis and binding studies of multiple calix[4]arenes

Abstract As novel host molecules, various double and quadruple calix[4]arenes have been synthesized by using quadruple cycloadditive macrocyclization, Schiff-base formation, and acylation. The interesting features of ‘head-to-head’ type multiple calix[4]arenes, such as the conformational aspects and cooperative binding, have been studied.

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]

Bis-calix[4]arenes with imine linkages: synthesis and binding study of thiopheno bis-calix[4]arene with viologens

Abstract We have developed an efficient synthetic pathway for various bis-calix[4]arenes with imine linkages by using a simple condensation procedure and carried out the binding study of thiopheno bis-calix[4]arene with biologically interesting viologen guests.

The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation

In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.