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WIREs Nanomed Nanobiotechnol
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Integration of chemically modified nucleotides with DNA strand displacement reactions for applications in living systems

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Abstract Watson–Crick base pairing rules provide a powerful approach for engineering DNA‐based nanodevices with programmable and predictable behaviors. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation. To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. In this review, we summarize recent progress toward the integration of chemically modified nucleotides with DNA strand displacement reactions, highlighting key successes in the development of robust systems and devices that operate in living cells and in vivo. We discuss the advantages and disadvantages of commonly employed modifications as they pertain to DNA strand displacement, as well as considerations that must be taken into account when applying modified oligonucleotide to living cells. Finally, we explore how chemically modified nucleotides fit into the broader goal of bringing dynamic DNA nanotechnology into the cell, and the challenges that remain. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing
Toehold‐mediated strand displacement reactions. (a) DNA strand displacement via three‐way branch migration. DNA is depicted as lines with the half arrow indicating the 3′‐end. A substrate strand having a single‐stranded toehold domain (t*) and a branch migration domain (a*) is hybridized to an incumbent strand (OUT) to form duplex A. Strand displacement is initiated through binding of the invader strand (IN) to the toehold domain of A (via t/t*) followed by a three‐way branch migration process in which domain a on the invading strand displaces domain a on the incumbent strand through a series of reversible dissociation/hybridization events. The reaction is complete once the invader strand (IN) fully displaces the incumbent strand (OUT) from duplex A. Toeholds accelerate the rate of strand displacement by increasing the probability that the incumbent strand is successfully replaced by the invader once bound. (b) DNA strand displacement via four‐way branch migration. Two DNA duplexes (IN1 and IN2) bind to each other through a pair of complementary toehold domains (t1/t1* and t2/t2*) to form a Holliday junction. A four‐way branch migration process ensues, resulting in the formation of two new DNA duplexes (OUT1 and OUT2) having more base pairs than the initial DNA duplexes
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Overview of the photocaging strategy for light‐mediated activation of DNA strand displacement reactions
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Intracellular strand displacement systems involving l‐DNA/RNA. (a) Schematic illustration of d‐DNA and l‐DNA reporter complexes (D/l‐R) (left) and their activation by specific and nonspecific pathways in cells (right). (b) Schematic illustration of the strand displacement cascade (left). Components for both the l‐DNA (heterochiral) and d‐DNA reaction cascades were transfected into cells and the reaction was initiated by UV treatment. Fluorescence was monitored by flow cytometry in the absence and presence of UV (right). Panels (a) and (b) were reprinted with permission from Zhong and Sczepanski (2021). Copyright 2021. American Chemical Society. (c) A fluorescent l‐RNA biosensor for imaging microRNA in living cells. Reprinted with permission from Zhong and Sczepanski (2019). Copyright 2019. American Chemical Society
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Strategies to sequence‐specifically interface d‐ and l‐oligonucleotides. (a) Heterochiral strand displacement using PNA–DNA heteroduplexes. (b) Heterochiral strand displacement using a single chimeric D/l‐DNA duplex. (c) Heterochiral strand displacement cascade using multiple chimeric D/l‐DNA duplexes. In this scheme, nuclease degradation of the d‐DNA portions of duplexes T1 and T2 will not result in the spurious release of the l‐DNA output, thereby avoiding reaction leak. Reprinted with permission from Mallette et al. (2020). Copyright 2020. American Chemical Society
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Intracellular strand displacement systems involving PNA. (a) Schematic representation of the nanoparticle‐mediated delivery of PNA strand displacement probes for imaging mRNA. Top: A quenched PNA–DNA duplex is electrostatically bound to the cationic nanoparticle and delivered into the cell. Upon entry and endosomal escape, binding of the PNA toehold to the target mRNA facilitates strand displacement of the quencher strand. Bottom: A pair of PNA–DNA FRET probes are delivered as above. Binding of each PNA toehold to the target mRNA facilitates strand displacement of the DNA strand. A FRET signal is observed only if both the donor and acceptor probes bind the mRNA. (b) Photocleavable PNA probe for gene knockdown in zebrafish. Photolysis‐induced cleavage of the photocleavable linker (PL) enables the probe to efficiently bind its target mRNA, resulting in the displacement of the blocking strand and arrest of protein synthesis by the PNA strand. Reprinted with permission from Tang et al. (2007). Copyright 2007. American Chemical Society. (c) PNA tagging of proteins (cell surface receptors in this case) provides a platform for recruitment of DNA strands for reversible fluorescent labeling by toehold‐mediated strand displacement. Reprinted with permission from Gavins et al. (2021). Copyright 2021. Springer Nature
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Intracellular Strand displacement systems involving 2′‐OMe modifications. (a) A four‐way strand exchange reaction designed to generate an active siRNA duplex in living cells, leading to gene silencing. Reprinted with permission from Groves et al. (2016). Copyright 2016. Springer Nature. (b) Strand displacement probe for imaging mRNA in living cells. Upon electroporation into cells, the probe binds the target mRNA resulting in displacement of the quencher strand. Co‐localization of multiple probes on the same mRNA allows single molecule‐level imaging. Reprinted with permission from Chatterjee et al. (2018). Copyright 2018. American Chemical Society
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Strand displacement systems involving PS modifications. (a) The strand displacement reaction with four‐way branch migration employed by the Seelig group. (b) Boolean AND and OR gates based on strand displacement via four‐way branch migration. The OR gate requires either Input A or Input B to activate fluorescence, whereas the AND gate requires both. Panels (a) and (b) are reprinted with permission from Groves et al., 2016. Copyright. Springer Nature. (c) Schematic illustration of a cascaded HCR device. A YES logic gate is shown. Hairpin HA is activated (exposure of domain 1*) upon binding to the microRNA target. The activated hairpin initiates an HCR cascade leading to fluorescence signal generation and amplification. Reprinted with permission from Gong et al. (2019). Copyright 2019. The Royal Society of Chemistry
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Schematic illustration of the hairpin DNA cascade amplifier (HDCA). The reporter complex was stabilized by the incorporation of LNAs. Reprinted with permission from Wu et al. (2015). Copyright 2015. American Chemical Society
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Diagnostic Tools > Biosensing
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Diagnostic Tools > In Vivo Nanodiagnostics and Imaging

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