Kevin M. Guckian

Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication

The physical and chemical factors that allow DNA to perform its functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of DNA have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving DNA has been the use of DNA base analogues in which functional groups are added, deleted, blocked, or rearranged. Here we describe a different strategy, in which the polar natural DNA bases are replaced by nonpolar aromatic molecules of the same size and shape. This allows the evaluation of polar interactions (such as hydrogen bonding) with little or no interference from steric effects. We have used these nonpolar nucleoside isosteres as probes of noncovalent interactions such as DNA base pairing and protein - DNA recognition. We have found that, while base-pairing selectivity does depend on Watson - Crick hydrogen bonding in the natural pairs, it is possible to design new bases that pair selectively and stably in the absence of hydrogen bonds. In addition, studies have been carried out with DNA polymerase enzymes to investigate the importance of Watson - Crick hydrogen bonding in enzymatic DNA replication. Surprisingly, this hydrogen bonding is not necessary for efficient enzymatic synthesis of a base pair, and significant levels of selectivity can arise from steric effects alone. These results may have significant impact both on the study of basic biomolecular interactions and in the design of new, functionally active biomolecules.

Solution structure of a DNA duplex containing a replicable difluorotoluene-adenine pair.

A nonpolar aromatic nucleoside derivative based on 2,4-difluorotoluene (F), a non-hydrogen bonding shape analog of thymidine, was recently shown to be replicated against adenine with high efficiency and fidelity. This led to the suggestion that geometric matching, potentially even in the absence of hydrogen bonding between bases in a pair, may be sufficient to direct nucleotide selection during replication. We have examined the solution structure of the F–A pair in the context of a 12 base pair DNA duplex. We find that, despite the destabilization caused by this analog, the F–A pair very closely resembles that of a T·A pair in the same context. This lends support to the importance of shape matching in replication.

Sequence‐Addressable DNA Logic

The creation of molecular Boolean logic gates is a crucial requirement in the quest to build molecular-scale computers. A variety of molecular logic gates have been devised previously based on designed organic molecules and peptides, deoxyribozymes, enzymatic biochemical networks, and biopolymer–ligand and photonic interactions. However, unlike electronic systems that share a common input/output (I/O) signal (the electron), I/O incompatibilities of molecular-scale logic gates often hamper their potential for assembly into higher-order circuits. In a step towards addressing this limitation, recent solutionand solid-phase approaches have exploited the inherent sequence specificity of DNA hybridization and strand-displacement events to direct communication between different logic gates. Herein, we present a series of entirely DNA-based logic gates (AND, OR, and XOR) that operate with mutually compatible oligonucleotide inputs and outputs. A DNA-based half-adder was assembled through the combination of an AND gate and an XOR (eXclusive OR) gate in a shared solution, thus demonstrating both the sequence addressability and cross-compatibility of the gates. The use of a single molecular-recognition platform gives these gates the potential to be assembled into higher-order circuits that are ‘‘virtually wired’’ through the information contained within oligonucleotide sequences. To demonstrate this potential, we ‘‘wired’’ two XOR gates into an OR gate and verified basic communication between these gates. Our approach harnesses the ability of single-stranded DNA to invade and displace shorter sections of complementary double-stranded DNA. Input oligonucleotides first bind short single-stranded ‘‘toehold’’ regions and then proceed to replace the base pairing in the smaller duplex via a three-way branch migration mechanism. The toehold region also allows the input DNA to form a longer duplex than the output strand, which provides the thermodynamic driving force for displacement. This approach was used to create an AND