Computationally reconstructing cotranscriptional RNA folding from experimental data reveals rearrangement of non-native folding intermediates.

The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.

Parallel molecular computation on digital data stored in DNA.

DNA is an incredibly dense storage medium for digital data. However, computing on the stored information is expensive and slow, requiring rounds of sequencing, in silico computation, and DNA synthesis. Prior work on accessing and modifying data using DNA hybridization or enzymatic reactions had limited computation capabilities. Inspired by the computational power of "DNA strand displacement," we augment DNA storage with "in-memory" molecular computation using strand displacement reactions to algorithmically modify data in a parallel manner. We show programs for binary counting and Turing universal cellular automaton Rule 110, the latter of which is, in principle, capable of implementing any computer algorithm. Information is stored in the nicks of DNA, and a secondary sequence-level encoding allows high-throughput sequencing-based readout. We conducted multiple rounds of computation on 4-bit data registers, as well as random access of data (selective access and erasure). We demonstrate that large strand displacement cascades with 244 distinct strand exchanges (sequential and in parallel) can use naturally occurring DNA sequence from M13 bacteriophage without stringent sequence design, which has the potential to improve the scale of computation and decrease cost. Our work merges DNA storage and DNA computing, setting the foundation of entirely molecular algorithms for parallel manipulation of digital information preserved in DNA.

DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes.

The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

Time-Controlled Authentication Strategies for Molecular Information Transfer.

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection.

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.

Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5 min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20 × 106 to 1.75 × 107  CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

Palindrome sequence mediated target recycling integrating with self-priming assisted signal reaction for sensitive miRNA detection.

The development of a sensitive and isothermal technique with a greatly enhanced miRNA detection signal is still technically problematic due to the low abundance of miRNA and high sequence similarities with homologous miRNAs. Herein, we propose a novel fluorescence approach for sensitive and reliable miRNA detection by integrating the palindrome sequence mediated target recycling with self-priming assisted signal reaction. In this method, a dual toehold DNA nano-probe (HT) with two functional arms is designed to mediate specific target recognition and signal amplification. In the presence of target miRNA, it binds to the recognition module of HT probe, releasing the "2" sequence to initiate strand displacement amplification (SDA) and a self-priming-induced signal reaction. Based on the elegant design, the proposed method exhibits a wide linear response range exceeding five orders of magnitude and a low limit of detection of 0.96 fM according to the 3δ rule. The non-specific signal is below 5 % for non-target miRNA detection. Taking the merits of excellent sensitivity, desirable specificity, and superior anti-interference ability, the proposed approach shows a promising prospect for detecting miRNAs in complicated biological environments and early diagnosis of diseases.

Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

2,6-diaminopurine (Z)-containing toehold probes improve genotyping sensitivity.

2,6-diaminopurine (Z), a naturally occurring noncanonical nucleotide base found in bacteriophages, enhances DNA hybridization by forming three hydrogen bonds with thymine (T). These distinct biochemical characteristics make it particularly valuable in applications that rely on the thermodynamics of DNA hybridization. However, the practical use of Z-containing oligos is limited by their high production cost and the challenges associated with their synthesis. Here, we developed an efficient and cost-effective approach to synthesize Z-containing oligos of high quality based on an isothermal strand displacement reaction. These newly synthesized Z-oligos are then employed as toehold-blockers in an isothermal genotyping assay designed to detect rare single nucleotide variations (SNV). When compared with their counterparts containing the standard adenine (A) base, the Z-containing blockers significantly enhance the accuracy of identifying SNV. Overall, our innovative methodology in the synthesis of Z-containing oligos, which can also be used to incorporate other unconventional and unnatural bases into oligonucleotides, is anticipated to be adopted for diverse applications, including genotyping, biosensing, and gene therapy.

Fluorescence biosensor to detect microRNAs via integrating DNA hairpins transition mediated strand displacement amplification with primer exchange reaction.

Herein, we constructed a fluorescence biosensor for the ultra-sensitive analysis of microRNAs (miRNAs) by combining DNA hairpins transition triggered strand displacement amplification (DHT-SDA) with primer exchange reaction (PER). Target miRNA initiated DHT-SDA to facilitate the generation of multiple single-stranded DNA (ssDNA) as PER primer, which was extended into a long ssDNA. The biosensor is successfully utilized in detecting miRNAs with high sensitivity (limit of detection for miRNA-21 was 58 fM) and a good linear relationship between 100 nM and 100 fM. By simply changing the DNA hairpin sequence, the constructed biosensor can be extended to analyze another miRNAs. Moreover, the biosensor has the feasibility of detecting miRNAs in real samples with satisfactory accuracy and reliability. Therefore, the fluorescent biosensor has great application potential in clinical diagnosis.

A target-triggered strand displacement-assisted target recycling based on carbon dots-based fluorescent probe and MSNs@PDA nanoparticles for miRNA amplified detection and fluorescence imaging.

A target-triggered strand displacement-assisted target recycling based on carbon dots-based fluorescent probe and mesoporous silica nanoparticles@polydopamine (MSNs@PDA) was established to detect miRNA. The surface of MSNs rich in mesopores was coated with a layer of PDA, which can adsorb and quench the fluorescence of single-stranded Fuel DNA with fluorescent carbon dots (CDs) modified at the end through fluorescence resonance energy transfer (FRET). After adding double-stranded DNA-gold nanoparticles (dsDNA-AuNPs) and target let-7a, it will trigger two toehold-mediated strand displacement reactions (TSDR), leading to the recovery of fluorescence and the recycling of target let-7a (excitation wavelength: 380 nm; emission wavelength: 458 nm). The recovery value of fluorescence is proportional to the logarithm of the target microRNA let-7a concentration, thus realizing the sensitivity amplification detection of disease markers. The MSNs@PDA@Fuel DNA-CDs/dsDNA-AuNPs nanoplatform based on the strategy of "on-off-on" and TSDR cyclic amplification may hold great potential as an effective and safe nanoprobe for accurate fluorescence imaging of diseases related to miRNA with low abundances.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Transient Dynamic Operation of G-Quadruplex-Gated Glucose Oxidase-Loaded ZIF-90 Metal-Organic Framework Nanoparticle Bioreactors.

Glucose oxidase-loaded ZIF-90 metal-organic framework nanoparticles conjugated to hemin-G-quadruplexes act as functional bioreactor hybrids operating transient dissipative biocatalytic cascaded transformations consisting of the glucose-driven H2O2-mediated oxidation of Amplex-Red to resorufin or the glucose-driven generation of chemiluminescence by the H2O2-mediated oxidation of luminol. One system involves the fueled activation of a reaction module leading to the temporal formation and depletion of the bioreactor conjugate operating the nickase-guided transient biocatalytic cascades. The second system demonstrates the fueled activation of a reaction module yielding a bioreactor conjugate operating the exonuclease III-dictated transient operation of the two biocatalytic cascades. The temporal operations of the bioreactor circuits are accompanied by kinetic models and computational simulations enabling us to predict the dynamic behavior of the systems subjected to different auxiliary conditions.

Environmentally Controlled Oscillator with Triplex Guided Displacement of DNA Duplexes.

The use of DNA triplex association is advantageous for the reconfiguration of dynamic DNA nanostructures through pH alteration and can provide environmental control for both structural changes and molecular signaling. The combination of pH-induced triplex-forming oligonucleotide (TFOs) binding with toehold-mediated strand displacement has recently garnered significant attention in the field of structural DNA nanotechnology. While most previous studies use single-stranded DNA to displace or replace TFOs within the triplex, here we demonstrate that pH alteration allows a DNA duplex, with a toehold assistance, to displace TFOs from the components of another DNA duplex. We examined the dependence of this process on toehold length and show that the pH changes allow for cyclic oscillations between two molecular formations. We implemented the duplex/triplex design onto the surface of 2D DNA origami in the form outlining binary digits 0 or 1 and verified the oscillatory conformational changes between the two formations with atomic force microscopy.

pH-Controlled Resettable Modular DNA Strand-Displacement Circuits.

Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

Kinetics of Strand Displacement Reaction with Acyclic Artificial Nucleic Acids.

Toehold-mediated strand displacement (TMSD) reaction, one of the DNA nanotechnologies, has great potential as s biological programmable platform in the cellular environment. Various artificial nucleic acids have been developed to improve stability and affinity for biological applications. However, the lack of understanding of the kinetics of TMSD reaction among artificial nucleic acids has limited their applications. We herein systematically characterized the kinetics of TMSD reactions with acyclic xeno nucleic acids (XNAs): serinol nucleic acid (SNA), acyclic D-threoninol nucleic acid (D-aTNA), and acyclic L-threoninol nucleic acid (L-aTNA). We found that the strand displacement reactions by D-aTNA and by L-aTNA were highly dependent on temperature. D-aTNA and L-aTNA systems were orthogonal to each other, and chirality of the input can be switched by using SNA as an interface. We also applied TMSD reactions of XNAs to a seesaw gate amplification system which utilizes the orthogonality. This work will contribute to the developments of thermoresponsive and bioorthogonal nucleic acid circuits.

PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.

The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.

Programming of Supercrystals Using Replicable DNA-Functionalized Colloids.

The development of self-replicating systems is of great importance in research on the origin of life. As the most iconic molecules, nucleic acids have provided prominent examples of the fabrication of self-replicating artificial nanostructures. However, it is still challenging to construct sophisticated synthetic systems that can create large-scale or three-dimensionally ordered nanomaterials using self-replicating nanostructures. By integrating a template system containing DNA-functionalized colloidal seeds with a simplified DNA strand-displacement circuit programmed subsystem to produce DNA-functionalized colloidal copies, we developed a facile enthalpy-mediated strategy to control the replication and catalytic assembly of DNA-functionalized colloids in a time-dependent manner. The replication efficiency and crystal quality of the resulting superlattice structures can be effectively increased by regulating the molar ratio of the template to the copy colloids. By constructing binary systems from two types of gold nanoparticles (or proteins), superlattice structures with different crystal symmetries can be obtained through the replication and catalytic assembly processes. This programmable enthalpy-mediated approach was easily leveraged to achieve the phase transformation and catalytic amplification of colloidal crystals starting from different initial template crystals. This work offers a potential way to construct self-replicating artificial systems that exhibit complicated phase behaviors and can produce large-scale superlattice nanomaterials.

A Cationic Copolymer Enhances Responsiveness and Robustness of DNA Circuits.

Toehold-mediated DNA circuits are extensively employed to construct diverse DNA nanodevices and signal amplifiers. However, operations of these circuits are slow and highly susceptive to molecular noise such as the interference from bystander DNA strands. Herein, this work investigates the effects of a series of cationic copolymers on DNA catalytic hairpin assembly, a representative toehold-mediated DNA circuit. One copolymer, poly(L -lysine)-graft-dextran, significantly enhances the reaction rate by 30-fold due to its electrostatic interaction with DNA. Moreover, the copolymer considerably alleviates the circuit's dependency on the length and GC content of toehold, thereby enhancing the robustness of circuit operation against molecular noise. The general effectiveness of poly(L -lysine)-graft-dextran is demonstrated through kinetic characterization of a DNA AND logic circuit. Therefore, use of a cationic copolymer is a versatile and efficient approach to enhance the operation rate and robustness of toehold-mediated DNA circuits, paving the way for more flexible design and broader application.