Heterochiral modifications enhance robustness and function of DNA in living human cells.

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactions in situ via a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror-image, ʟ-DNA nucleotides to produce heterochiral "gapmers". We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ-nucleic acid protection of oligonucleotides and DNA circuitry for applications in vivo.

Protecting Heterochiral DNA Nanostructures against Exonuclease-Mediated Degradation.

Heterochiral DNA nanotechnology employs nucleic acids of both chiralities to construct nanoscale devices for applications in the intracellular environment. Interacting directly with cellular nucleic acids can be done most easily using D-DNA of the naturally occurring right-handed chirality; however, D-DNA is more vulnerable to degradation than enantiometric left-handed L-DNA. Here we report a novel combination of D-DNA and L-DNA nucleotides in triblock heterochiral copolymers, where the L-DNA domains act as protective caps on D-DNA domains. We demonstrate that the D-DNA components of strand displacement-based molecular circuits constructed using this technique resist exonuclease-mediated degradation during extended incubations in serum-supplemented media more readily than similar devices without the L-DNA caps. We show that this protection can be applied to both double-stranded and single-stranded circuit components. Our work enhances the state of the art for robust heterochiral circuit design and could lead to practical applications such as in vivo biomedical diagnostics.

Robust Heterochiral Strand Displacement Using Leakless Translators.

Molecular computing offers a powerful framework for in situ biosensing and signal processing at the nanoscale. However, for in vivo applications, the use of conventional DNA components can lead to false positive signals being generated due to degradation of circuit components by nuclease enzymes. Here, we use hybrid chiral molecules, consisting of both l- and d-nucleic acid domains, to implement leakless signal translators that enable d-nucleic acid signals to be detected by hybridization and then translated into a robust l-DNA signal for further analysis. We show that our system is robust to false positive signals even if the d-DNA components are degraded by nucleases, thanks to circuit-level robustness. This work thus broadens the scope and applicability of DNA-based molecular computers for practical, in vivo applications.