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Classically, a molecular element (ME) is a pure substance composed of two or more atoms of the same element. However, MEs, in the context of this review, can be any molecules as elements bonded together into the backbone of synthetic oligonucleotides (ONs) with designed sequences and functions, including natural A, T, C, G, U, and unnatural bases. The use of MEs can facilitate the synthesis of designer molecules and smart materials. In particular, we discuss the landmarks associated with DNA structure and related technologies, as well as the extensive application of ONs, the ideal type of molecules for intervention therapy aimed at correcting disease-causing genetic errors (indels). It is herein concluded that the discovery of ON therapeutics and the fabrication of designer molecules or nanostructures depend on the ME concept that we previously published. Accordingly, ME will be our focal point as we discuss related research directions and perspectives in making molecules and materials. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

The design of controllable dynamic systems is vital for the construction of organelle-like architectures in living cells, but has proven difficult due to the lack of control over defined topological transformation of self-assembled structures. Herein, we report a DNA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle-like hydrogel architecture in living cells. Designer DNA nanoparticles are constructed from double-stranded DNA with cytosine-rich stick ends (C-monomer) and are internalized into cells through lysosomal pathway. The lysosomal acidic microenvironment can activate the assembly of DNA monomers, inducing transformation from nanoparticles to micro-sized organelle-like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors: cytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted., (© 2020 Wiley-VCH GmbH.)

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

Phosphorylation of DNA by polynucleotide kinase (PNK) takes an important role in DNA damage repair, replication and recombination. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, we present a label-free fluorescent method for PNK activity assay using double strand DNA (dsDNA) -templated copper nanoparticles (CuNPs) as a fluorescent indicator. Upon the PNK reaction, the dsDNA template is phosphorylated and then digested by λ exonuclease immediately, prohibiting the formation of fluorescent CuNPs due to the lack of dsDNA template. This homogeneous PNK activity assay does not require any other additional modifications of DNA substrate or complex design, making the proposed strategy simple, cost-effective and high throughput. The proposed strategy is sensitive, selective and exhibits a good assay performance in complex biological samples. The strategy presented here opens a new avenue for PNK assay and nucleic acid phosphorylation related research., (Copyright © 2012 Elsevier B.V. All rights reserved.)

T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5'-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked 'HRPzyme' sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL(-1) and a wide detection range from 0.06 to 100 U mL(-1). Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Based on a target-activated autocatalytic DNAzyme amplification strategy, novel fluorescence sensing platforms were constructed for highly sensitive and selective assay of base excision repair enzyme activity. By using a rolling circle amplification (RCA)-coupled amplification cascade, an extremely low detection limit (0.002 U mL(-1)) was achieved.

A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs., (This journal is © The Royal Society of Chemistry 2012)

A novel electrochemical strategy that uses DNA-wrapped carbon nanotubes (CNTs) as electrochemical labels is developed for sensitive and selective detection of sequence-specific DNA. The presence of target DNA mediates the formation of a sandwiched complex between the DNA-wrapped CNT and a hairpin DNA capture probe immobilized on magnetic beads. This allows target-selective collection of the CNT labels by magnetic separation and transfer on the electrode surface modified with an insulating self-assembled monolayer (SAM). After treatment with N,N-dimethylformamide, the collected sandwiched complex releases the bare CNTs and facilitates the removal of magnetic beads from the electrode surface. The bare CNTs can then assemble on the SAM-modified electrode surface and mediate efficient electron transfer between the electrode and the electroactive species in the solution with a strong current signal generated. The results indicate that the developed strategy shows a sensitive response to target DNA with a desirable signal gain and a low detection limit of 0.9 pM. This strategy is also demonstrated to provide excellent differentiation of single-base mismatch in target DNA. It is expected that this electrochemical strategy may hold great potential as a novel platform for clinical diagnostics and genetic analysis., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This paper describes a novel electrochemical DNA sensor for the simple, sensitive and specific detection of nucleic acids based on proximity-dependent DNA ligation assays with the DNAzyme amplification of hairpin substrate signal. A long DNA strand contains the catalytic motif of Mg(2+)-dependent 10-23 DNAzyme, acting as the recognition probe. When the target DNA was introduced into the system, part of it was complementary to 5'-end of the recognition probe, resulting in the ligation of a stable duplex, the unbinded part of the target DNA was acted as one binding arm for the DNAzyme. This duplex containing a complete 10-23 DNAzyme structure could cleave the purine-pyrimidine cleavage site of the hairpin substrate, which resulted in the fragmentation of the hairpin structure and the release of two single-stranded nucleic acids, one of which was biotinylated and acted as the signal probe. An immobilized thiolated capture probe could bind with the signal probe, using biotin as a tracer in the signal probe, and streptavidin-alkaline phosphatase (SA-ALP) as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 5 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The results revealed that the sensor showed a sensitive response to complementary target sequences of H. pylori in a concentration range from 100 fM to 1 nM, with a detection limit of 50 fM. In addition, the sensing system could discriminate the complementary sequence from mismatched sequences, with high sensitivity and reusability., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

We described a novel electrochemical DNA biosensor based on molecular beacon (MB) probe and enzymatic amplification protocol. The MB modified with a thiol at its 5' end and a biotin at its 3' end was immobilized on the gold electrode through mixed self-assembly process. Hybridization events between MB and target DNA cause the conformational change of the MB, triggering the attached biotin group on the electrode surface. Following the specific interaction between the conformation-triggered biotin and streptavidin-horseradish peroxidase (HRP), subsequent quantification of DNA was realized by electrochemical detection of enzymatic product in the presence of substrate. The detection limit is obtained as low as 0.1nM. The presented DNA biosensor has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing G-G single-base mismatches.

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.

DNA logic circuits (DLC) enable the accurate identification of specific cell types, such as cancer cells, but they face the challenges of weak output signals and a lack of competent platforms that can efficiently deliver DLC components to the target site in the living body. To address these issues, we rationally introduced a cascaded biological amplifier module based on the Primer Exchange Reaction inspired by electronic circuit amplifier devices. As a paradigm, three abnormally expressed Hela cell microRNAs (‐30a, ‐17, and ‐21) were chosen as "AND" gate inputs. DLC response to these inputs was boosted by the amplifier markedly enhancing the output signal. More importantly, the encapsulation of DLC and amplifier components into ZIF‐8 nanoparticles resulted in their efficient delivery to the target site, successfully distinguishing the Hela tumor subtype from other tumors in vivo. Thus, we envision that this strategy has great potential for clinical cancer diagnosis. [ABSTRACT FROM AUTHOR]

The design of controllable dynamic systems is vital for the construction of organelle‐like architectures in living cells, but has proven difficult due to the lack of control over defined topological transformation of self‐assembled structures. Herein, we report a DNA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle‐like hydrogel architecture in living cells. Designer DNA nanoparticles are constructed from double‐stranded DNA with cytosine‐rich stick ends (C‐monomer) and are internalized into cells through lysosomal pathway. The lysosomal acidic microenvironment can activate the assembly of DNA monomers, inducing transformation from nanoparticles to micro‐sized organelle‐like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors: cytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted. [ABSTRACT FROM AUTHOR]

A novel label-free fluorescent strategy for the detection of glutathione (GSH) and cysteine (Cys) is presented. The system consists of two single stranded DNA (ssDNA) with thymine–thymine (T–T) mismatches and used Hg2+ as a mediator, and N-methyl mesoporphyrin IX (NMM) as the signal reporter. The assay is based on the competitive reaction of Hg2+ with GSH/Cys and T–T mismatched double stranded DNA (dsDNA). In the absence of the target, two ssDNA containing T–T mismatches react with Hg2+ to form a T–Hg2+–T dsDNA structure in the solution, which hampers the formation of a G-quadruplex structure. However, in the presence of the target, GSH/Cys reacts with Hg2+ to keep DNA probes in a free single state, resulting in the effective formation of a G-quadruplex structure of the DNA probe (GP). Subsequently, due to the strong interaction between the G-quadruplex structure and NMM, fluorescence was greatly enhanced. This fluorescence strategy does not require any chemical modification, making the assay convenient and cost-effective. This method exhibited a linear relationship between peak fluorescence intensity and concentration of GSH in the range of 10–400 nM with a limit of detection (LOD) of 9.6 nM. A linear range for Cys detection was obtained in the concentration range of 10–500 nM with an LOD of 10 nM. Moreover, the proposed method worked well for the analysis of complex biological samples. [ABSTRACT FROM AUTHOR]

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported. [ABSTRACT FROM AUTHOR]

Abstract: A novel electrochemical aptasensor based on hybridization chain reaction (HCR) with enzyme-signal amplification was constructed for the detection of interferon-gamma (IFN-γ). In this aptasensor, the recognition probes which contained the sequence of IFN-γ aptamer were initially binded to IFN-γ, and the unbound recognition probes were captured on the electrode as an initiator to trigger the HCR. The two DNA hairpins bio-H1 and bio-H2 were opened by the recognition probe, and bound one by one on the electrode. The biotin was used as a tracer in the hairpins and streptavidin–alkaline phosphatase (SA–ALP) as a reporter molecule. Then, SA–ALP converted its electro-inactive substrate 1-naphthyl phosphate into an electroactive derivative 1-naphthol generating amplified electrochemical signal by differential pulse voltammetry (DPV). The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated for enzymatic dephosphorylation of 1-naphthyl phosphate. The electrochemical signal observed was inversely related to the concentration of IFN-γ. The proposed approach showed a high sensitivity for IFN-γ in a concentration range of 0.5–300nM with a detection limit of 0.3nM. The sensing system also provided satisfactory results for the detection of IFN-γ in the cell media. [Copyright &y& Elsevier]