H. Tom Soh, Postdoctoral Faculty Sponsor
Heterochiral DNA strand-displacement reactions enable sequence-specific interfacing of oligonucleotide enantiomers, making it possible to interface native d-nucleic acids with molecular circuits built using nuclease-resistant l-DNA. To date, all heterochiral reactions have relied on peptide nucleic acid (PNA), which places potential limits on the scope and utility of this approach. Herein, we now report heterochiral strand-displacement in the absence of PNA, instead utilizing chimeric d/l-DNA complexes to interface oligonucleotides of the opposite chirality. We show that these strand-displacement reactions can be easily integrated into multicomponent heterochiral circuits, are compatible with both DNA and RNA inputs, and can be engineered to function in serum-supplemented medium. We anticipate that these new reactions will lead to a wider application of heterochiral strand-displacement, especially in the design of biocompatible nucleic acid circuits that can reliably operate within living systems.
View details for DOI 10.1021/acssynbio.9b00335
View details for Web of Science ID 000504805800017
View details for PubMedID 31670930
View details for PubMedCentralID PMC6953401
The programmability of DNA/RNA-based molecular circuits provides numerous opportunities in the field of synthetic biology. However, the stability of nucleic acids remains a major concern when performing complex computations in biological environments. Our solution to this problem is L-(deoxy)ribose nucleic acids (L-DNA/RNA), which are mirror images (i.e. enantiomers) of natural D-nucleotides. L-oligonucleotides have the same physical and chemical properties as their natural counterparts, yet they are completely invisible to the stereospecific environment of biology. We recently reported a novel strand-displacement methodology for transferring sequence information between oligonucleotide enantiomers (which are incapable of base pairing with each other), enabling bio-orthogonal L-DNA/RNA circuits to be easily interfaced with living systems. In this perspective, we summarize these so-called "heterochiral" circuits, provide a viewpoint on their potential applications in synthetic biology, and discuss key problems that must be solved before achieving the ultimate goal of engineering complex and reliable functionality.
View details for DOI 10.1042/ETLS20190102
View details for Web of Science ID 000496157900009
View details for PubMedID 33501379
View details for PubMedCentralID PMC7832003
As chiral molecules, naturally occurring d-oligonucleotides have enantiomers, l-DNA and l-RNA, which are comprised of l-(deoxy)ribose sugars. These mirror-image oligonucleotides have the same physical and chemical properties as that of their native d-counterparts, yet are highly orthogonal to the stereospecific environment of biology. Consequently, l-oligonucleotides are resistant to nuclease degradation and many of the off-target interactions that plague traditional d-oligonucleotide-based technologies; thus making them ideal for biomedical applications. Despite a flurry of interest during the early 1990s, the inability of d- and l-oligonucleotides to form contiguous Watson-Crick base pairs with each other has ultimately led to the perception that l-oligonucleotides have only limited utility. Recently, however, scientists have begun to uncover novel strategies to harness the bio-orthogonality of l-oligonucleotides, while overcoming (and even exploiting) their inability to Watson-Crick base pair with the natural polymer. Herein, a brief history of l-oligonucleotide research is presented and emerging l-oligonucleotide-based technologies, as well as their applications in research and therapy, are presented.
View details for DOI 10.1002/chem.201900149
View details for Web of Science ID 000474807200003
View details for PubMedID 30913332
The absence of a straightforward strategy to interface native d-DNA with its enantiomer l-DNA-oligonucleotides of opposite chirality are incapable of forming contiguous Watson-Crick base pairs with each other-has enforced a "homochiral" paradigm over the field of dynamic DNA nanotechnology. As a result, chirality, a key intrinsic property of nucleic acids, is often overlooked as a design element for engineering of DNA-based devices, potentially limiting the types of behaviors that can be achieved using these systems. Here we introduce a toehold-mediated strand-displacement methodology for transferring information between orthogonal DNA enantiomers via an achiral intermediary, opening the door for "heterochiral" DNA nanotechnology having fully interfaced d-DNA and l-DNA components. Using this approach, we demonstrate several heterochiral DNA circuits having novel capabilities, including autonomous chiral inversion of DNA sequence information and chirality-based computing. In addition, we show that heterochiral circuits can directly interface endogenous RNAs (e.g., microRNAs) with bioorthogonal l-DNA, suggesting applications in bioengineering and nanomedicine. Overall, this work establishes chirality as a design parameter for engineering of dynamic DNA nanotechnology, thereby expanding the types of architectures and behaviors that can be realized using DNA.
View details for DOI 10.1021/jacs.7b10038
View details for Web of Science ID 000418204600004
View details for PubMedID 29182318