Modular Aptamer Switches for the Continuous Optical Detection of Small-Molecule Analytes in Complex Media.

Aptamers are a promising class of affinity reagents because signal transduction mechanisms can be built into the reagent, so that they can directly produce a physically measurable output signal upon target binding. However, endowing the signal transduction functionality into an aptamer remains a trial-and-error process that can compromise its affinity or specificity and typically requires knowledge of the ligand binding domain or its structure. In this work, a design architecture that can convert an existing aptamer into a "reversible aptamer switch" whose kinetic and thermodynamic properties can be tuned without a priori knowledge of the ligand binding domain or its structure is described. Finally, by combining these aptamer switches with evanescent-field-based optical detection hardware that minimizes sample autofluorescence, this study demonstrates the first optical biosensor system that can continuously measure multiple biomarkers (dopamine and cortisol) in complex samples (artificial cerebrospinal fluid and undiluted plasma) with second and subsecond-scale time responses at physiologically relevant concentration ranges.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as 'heterochiral' strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction - strand displacement from PNA-DNA heteroduplexes - remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA-DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA-DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Nuclease Hydrolysis Does Not Drive the Rapid Signaling Decay of DNA Aptamer-Based Electrochemical Sensors in Biological Fluids.

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 nuclease-resistant 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.

Heterochiral DNA Strand-Displacement Based on Chimeric d/l-Oligonucleotides.

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.

Mirror-Image Oligonucleotides: History and Emerging Applications.

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.

Heterochiral Nucleic Acid Circuits.

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.

Heterochiral DNA Strand-Displacement Circuits.

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.