Young Jun Seo

PCR with an expanded genetic alphabet.

Expansion of the genetic alphabet with a third base pair would lay the foundation for a semisynthetic organism with an expanded genetic code and also have immediate in vitro applications. Previously, the unnatural base pairs formed between d5SICS and either dNaM or dMMO2 were shown to be well-replicated by DNA polymerases under steady-state conditions and also transcribed by T7 RNA polymerase efficiently in either direction. We now demonstrate that DNA containing either the d5SICS-dNaM or d5SICS-dMMO2 unnatural base pair may be amplified by PCR with fidelities and efficiencies that approach those of fully natural DNA. These results further demonstrate that the determinants of a functional unnatural base pair may be designed into predominantly hydrophobic nucleobases with no structural similarity to the natural purines or pyrimidines. Importantly, the results reveal that the unnatural base pairs may function within an expanded genetic alphabet and make possible many in vitro applications.

Transcription of an Expanded Genetic Alphabet

Expansion of the genetic alphabet with a third base pair would have immediate biotechnology applications and also lay the foundation for a semisynthetic organism with an expanded genetic code. A variety of unnatural base pairs have been shown to be formed efficiently and selectively during DNA replication, and the pairs formed between the unnatural nucleotide d5SICS and either dMMO2 or dNaM are particularly interesting because they have been shown to be replicated with efficiencies and fidelities that are beginning to approach those of a natural base pair. Not only are these unnatural base pairs promising for different applications, but they also demonstrate that nucleobase shape and hydrophobicity are sufficient to control replication. While a variety of unnatural base pairs have been shown to be substrates for transcription, none are transcribed in both possible strand contexts, and the transcription of a fully hydrophobic base pair has not been demonstrated. We show here that both of the unnatural base pairs d5SICS:dMMO2 and d5SICS:dNaM are selectively transcribed by T7 RNA polymerase and that the efficiency of d5SICS:dNaM transcription in both possible strand contexts is only marginally reduced relative to that of a natural base pair. Thus, as with replication, we find that hydrogen-bonding is not essential for transcription and may be replaced with packing and hydrophobic forces. The results also demonstrate that d5SICS:dNaM is both replicated and transcribed with efficiencies and fidelities that should be sufficient for use as part of an in vitro expanded genetic alphabet.

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.

Site-Specific Labeling of DNA and RNA Using an Efficiently Replicated and Transcribed Class of Unnatural Base Pairs

Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.

Probing the B-to-Z-DNA duplex transition using terminally stacking ethynyl pyrene-modified adenosine and uridine bases

Pyrene-modified adenosine and uridine bases located in the dangling positions of G,C-alternating oligodeoxynucleotides undergo pi-stacking in their B-DNA duplexes, but not in their Z-DNA duplexes; fluorescence quenching in the former, through photoinduced electron transfer, but not in the latter, allows the state of the B-to-Z-DNA transition to be characterized visually.

Probing the stable G-quadruplex transition using quencher-free end-stacking ethynyl pyrene–adenosine

Pyrene-modified adenosines in the dangling positions of G-rich oligodeoxynucleotides undergo pi-stacking in their G-quadruplex formation, but not in their single strands, which can be characterized by fluorescence lambda(max) changes that occur on stacking.

Reversible sol-gel signaling system with epMB for the study of enzyme- and pH-triggered oligonucleotide release from a biotin hydrogel

The biotin-based low molecular weight hydrogel (G1) is able to entrap the epMB and as a consequence, the color of the epMB changes from green to blue; the color change depends on the state of the gelator, i.e. upon proceeding from sol to gel and vice versa.

Major Groove Derivatization of an Unnatural Base Pair

An unnatural base pair that is replicated and transcribed with good efficiency would lay the foundation for the long term goal of creating a semisynthetic organism, but also would have immediate in vitro applications, such as the enzymatic synthesis of site‐specifically modified DNA and/or RNA. One of the most promising of the unnatural base pairs that we have identified is formed between d5SICS and dMMO2. The ortho substituents of these nucleotides are included to facilitate unnatural base pair extension, presumably by forming a hydrogen‐bond with the polymerase, but the synthesis of the unnatural base pair still requires optimization. Recently, we have shown that meta and/or para substituents within the dMMO2 scaffold can facilitate unnatural base pair synthesis, although the mechanism remains unclear. To explore this issue, we synthesized and evaluated several dMMO2 derivatives with meta‐chlorine, ‐bromine, ‐iodine, ‐methyl, or ‐propinyl substituents. Complete characterization of unnatural base pair and mispair synthesis and extension reveal that the modifications have large effects only on the efficiency of unnatural base pair synthesis and that the effects likely result from a combination of changes in steric interactions, polarity, and polarizability. The results also suggest that functionalized versions of the propinyl moiety of d5PrM should serve as suitable linkers to site‐specifically incorporate other chemical functionalities into DNA. Similar modifications of d5SICS should allow labeling of DNA with two different functionalities, and the previously demonstrated efficient transcription of the unnatural base pair suggests that derivatives might similarly enable site‐specific labeling of RNA.

Detection of structure-switching in G-quadruplexes using end-stacking ability.

G-quadruplex-forming ODNs containing nonpolar aromatic fluorophore moiety, A(PY) at the dangling ends undergo pi-stacking on surface of G-quadruplex, and the fluorescence change can be used to distinguish the structure-switching between the mixed parallel/antiparallel structure and antiparallel structure.

Cholesterol-Linked Pyrene Excimer Molecular Beacon with Enhanced Cell Permeability

Covelently labeled pyrene excimer molecular beacon (MB) with cholesterol moiety has been developed for enhanced the cellular delivery of MB.(1) Pyrene units were covalently attached into adenosine and incorporated to oligonucleotides at the complementary locations in opposite strands in the middle positions of hairpin stems. The system behaves as an effective MB that changes color from green to blue upon duplex formation. A cholesterol unit was also attached into a free terminus of one of these hairpins. The cholesterol-linked MBs enhanced the cellular delivery of the MBs and showed similar cell permeability to conventional transfection methods. These structurally simple cholesterol-based MB systems, which can be synthesized very efficiently, have good potential for opening up new and exciting opportunities in the field of in vivo biosensors.