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.

Electrochemical aptamer-based (E-AB) sensors are a technology capable of real-time monitoring of drug concentrations directly in the body. These sensors achieve their selectivity from surface-attached aptamers, which alter their conformation upon target binding, thereby causing a change in electron transfer kinetics between aptamer-bound redox reporters and the electrode surface. Because, in theory, aptamers can be selected for nearly any target of interest, E-AB sensors have far-reaching potential for diagnostic and biomedical applications. However, a remaining critical weakness in the platform lies in the time-dependent, spontaneous degradation of the bioelectronic interface. This progressive degradationseen in part as a continuous drop in faradaic current from aptamer-attached redox reporterslimits the in vivo operational life of E-AB sensors to less than 12 h, prohibiting their long-term application for continuous molecular monitoring in humans. In this work, we study the effects of nuclease action on the signaling lifetime of E-AB sensors, to determine whether the progressive signal loss is caused by hydrolysis of DNA aptamers and thus the loss of signaling moieties from the sensor surface. We continuously interrogate sensors deployed in several undiluted biological fluids at 37 °C and inject nuclease to reach physiologically relevant concentrations. By employing both naturally occurring D-DNA and the nucleaseresistant enantiomer L-DNA, we determine that within the current lifespan of state-of-the-art E-AB sensors, nuclease hydrolysis is not the dominant cause of sensor signal loss under the conditions we tested. Instead, signal loss is driven primarily by the loss of monolayer elementsboth blocking alkanethiol and aptamer monolayersfrom the electrode surface. While use of L-DNA aptamers may extend the E-AB operational life in the long term, the critical issue of passive monolayer loss must be addressed before those effects can be seen.

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.

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.

Current technology for measuring specific biomarkers – continuously in complex samples, without sample preparation – is limited to just handful of molecules such as glucose and blood oxygen. In this work, we present the first optical biosensor system that enables continuous detection of a wide range of biomarkers in complex samples, such as human plasma. Our system employs a modular duplex-bubble switch (DBS) architecture that converts aptamers into structure-switching fluorescence probes whose affinity and kinetics can be readily tuned. These DBS constructs are coupled to a fiber-optic detector that measures the fluorescence change only within an evanescent field, thereby minimizing the impact of background autofluorescence and enabling direct detection of analytes at physiologically relevant concentrations even in interferent-rich sample matrices. Using our system, we achieved continuous detection of dopamine in artificial cerebrospinal fluid for >24 hours with sub-second resolution and a limit of detection (LOD) of 1 µM. We subsequently demonstrated the system’s generalizability by configuring it to detect cortisol with nanomolar sensitivity in undiluted human plasma. Both sensors achieved LODs orders of magnitude lower than theKDof the DBS element, highlighting the potential to achieve sensitive detection even when using aptamers with modest affinity.