Nuclease-Resistant L-DNA Tension Probes Enable Long-Term Force Mapping of Single Cells and Cell Consortia.

DNA-based tension probes with precisely programmableforce response provide important insights into cellularmechanosensing. However, their degradability in cell culture limitstheir use for long-term imaging, for instance, when cells migrate,divide, and differentiate. This is a critical limitation for providinginsights into mechanobiology for these longer-term processes. Here,we present DNA-based tension probes that are entirely designedbased on the stereoisomer of biological D-DNA, i.e., L-DNA. Wedemonstrate that L-DNA tension probes are essentially indestructibleby nucleases and provide days-long imaging without significant lossin image quality. We also show their superiority already for shortimaging times commonly used for classical D-DNA tension probes.We showcase the potential of these resilient probes to image minutemovements, and for generating long term force maps of single cellsand of collectively migrating cell populations.

A Biostable l-DNA Hydrogel with Improved Stability for Biomedical Applications.

DNA hydrogels have attracted increasing attention owing to their excellent permeability and high mechanical strength, together with thixotropy, versatile programmability and good biocompatibility. However, the moderate biostability and immune stimulation of DNA have arisen as big concerns for future potential clinical applications. Herein, we report the self-assembly of a novel l-DNA hydrogel, which inherited the extraordinary physical properties of a d-DNA hydrogel. With the mirror-isomer deoxyribose, this hydrogel exhibited improved biostability, withstanding fetal bovine serum (FBS) for at least 1 month without evident decay of its mechanical properties. The low inflammatory response of the l-DNA hydrogel has been verified both in vitro and in vivo. Hence, this l-DNA hydrogel with outstanding biostability and biocompatibility can be anticipated to serve as an ideal 3D cell-culture matrix and implanted bio-scaffold for long-term biomedical applications.

Identification of Cannabis Sativa L. Based on rbcL Sequence.

ABSTRACT: Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

Construction and Characterization of a Mirror-Image l-DNA i-Motif.

The first thermally stable and pH-responsive quadruplex intercalated motif (i-motif) structure formed by l-DNA is presented. Although this l-type i-motif exhibits the same physiochemical properties as its d isomer, its inverted chirality and good enzymatic resistance potentially open the way to the development of new DNA materials of pharmaceutical and biological interest.

l-DNA Duplex Formation as a Bioorthogonal Information Channel in Nucleic Acid-Based Surface Patterning.

Photolithographic in situ synthesis of nucleic acids enables extremely high oligonucleotide sequence density as well as complex surface patterning and combined spatial and molecular information encoding. No longer limited to DNA synthesis, the technique allows for total control of both chemical and Cartesian space organization on surfaces, suggesting that hybridization patterns can be used to encode, display or encrypt informative signals on multiple chemically orthogonal levels. Nevertheless, cross-hybridization reduces the available sequence space and limits information density. Here we introduce an additional, fully independent information channel in surface patterning with in situ l-DNA synthesis. The bioorthogonality of mirror-image DNA duplex formation prevents both cross-hybridization on chimeric l-/d-DNA microarrays and also results in enzymatic orthogonality, such as nuclease-proof DNA-based signatures on the surface. We show how chimeric l-/d-DNA hybridization can be used to create informative surface patterns including QR codes, highly counterfeiting resistant authenticity watermarks, and concealed messages within high-density d-DNA microarrays.

l-DNA-Based Catalytic Hairpin Assembly Circuit.

Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic hairpin assembly (CHA) circuit constructed from nuclease-resistant l-DNA that is capable of unimpeded signal amplification in the presence of 10% fetal bovine serum (FBS). The superior biostability of the l-DNA CHA circuit relative to its native d-DNA counterpart was clearly demonstrated through a direct comparison of the two systems (d versus l) under various conditions. Importantly, we show that the l-CHA circuit can be sequence-specifically interfaced with an endogenous d-nucleic acid biomarker via an achiral peptide nucleic acid (PNA) intermediary, enabling catalytic detection of the target in FBS. Overall, this work establishes a blueprint for the detection of low-abundance nucleic acids in harsh biological environments and provides further impetus for the construction of DNA nanotechnology using l-oligonucleotides.

Mirror-Image DNA Nanobox for Enhancing Environment Resistance of Nucleic Acid Probes.

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.

DNA vs. mirror-image DNA: a universal approach to tune the absolute configuration in DNA-based asymmetric catalysis.

Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test.

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

Addition of mirror-image L-DNA elements to DNA amplification circuits to distinguish leakage from target signal.

DNA amplification circuits that rely on thermodynamically-driven hybridization events triggered by a target nucleic acid are becoming increasingly utilized due to their relative simplicity. A drawback of these circuits is that non-specific amplification, or circuit leakage, must be estimated using a separate "no-target" control reaction to eliminate false positives. Aside from requiring an additional reaction, the problem with this approach is the difficulty of creating a no-target control for biological specimens. To overcome this limitation, we propose a strategy that combines both reactions into the same tube using naturally-occurring right-handed D-DNA circuit elements for the target detection reaction and identical synthetic mirror-image left-handed L-DNA circuit elements for the no-target control reaction. We illustrate this approach using catalyzed hairpin assembly (CHA), one of the most studied DNA amplification circuits. In a dual-chirality CHA design, the right-handed circuit signal is produced by target-specific amplification and circuit leakage, whereas the left-handed circuit signal is produced only by circuit leakage. The target-specific amplification is calculated as the difference between the two signals. The limit of detection of this dual-chirality CHA reaction was found to be similar to that of traditional CHA (81 vs 92 pM, respectively). Furthermore, the left-handed no-target signal matched the right-handed leakage across a wide range of sample conditions including background DNA, increased salt concentration, increased temperature, and urine. These results demonstrate the robustness of a dual-chirality design and the potential utility of left-handed DNA in the development of new DNA amplification circuits better-suited for target detection applications in biological samples.

Direct Comparison of d-DNA and l-DNA Strand-Displacement Reactions in Living Mammalian Cells.

To overcome technical challenges associated with the use of DNA strand-displacement circuits in vivo, including degradation by cellular nucleases, researchers are increasingly turning to bio-orthogonal l-DNA. Although enhanced stability and improved performance of l-DNA-based circuits within living cells are often implied, direct experimental evidence has not been provided. Herein, we directly compare the functional stability and kinetics of d-DNA and l-DNA strand-displacement in live cells for the first time. We show that l-DNA strand-displacement reaction systems have minimal "leak", fast reaction kinetics, and prolonged stability inside living cells as compared to conventional d-DNA. Furthermore, using "heterochiral" strand-displacement, we demonstrate that biostable l-DNA reaction components can be easily interfaced with native DNA inside cells. Overall, our results strongly support the broader adoption of l-DNA in the field of DNA molecular circuitry, especially for in vivo applications.

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase.

The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Its use is well established in applications aiming for extension of a DNA initiator strand, but a more recent focus points to its potential in enzymatic de novo synthesis of DNA. Whereas its low substrate specificity for nucleoside triphosphates has been studied extensively, here we interrogate how the activity of TdT is modulated by the nature of the initiating strands, in particular their length, chemistry, and nucleotide composition. Investigation of full permutational libraries of mono- to pentamers of d-DNA, l-DNA, and 2'O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence. We also show TdT being catalytically active on a non-nucleosidic substrate, hexaethylene glycol. These results offer new perspectives on constraints and strategies for de novo synthesis of DNA using TdT regarding the requirements for initiation of enzymatic generation of DNA.

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.

Sequencing Mirror-Image DNA Chemically.

The development of mirror-image biology systems faces a crucial barrier of lacking an L-DNA sequencing technique. Here, we developed a practical method for sequencing mirror-image DNA by adopting the Maxam-Gilbert sequencing approach, through which specific nucleobases in an end-labeled L-DNA are cleaved by achiral chemicals. This technique may facilitate the therapeutic application of nuclease-resistant L-aptamer drugs, and bring the vision of building an alternative, mirror-image self-replicating system closer to reality.

Mirror-image polymerase chain reaction.

The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.