Rolling circle amplification (RCA) is an isothermal enzymatic process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA template and special DNA or RNA polymerases. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. The power, simplicity, and versatility of the DNA amplification technique have made it an attractive tool for biomedical research and nanobiotechnology. Traditionally, RCA has been used to develop sensitive diagnostic methods for a variety of targets including nucleic acids (DNA, RNA), small molecules, proteins, and cells. RCA has also attracted significant attention in the field of nanotechnology and nanobiotechnology. The RCA-produced long, single-stranded DNA with repeating units has been used as template for the periodic assembly of nanospecies. Moreover, since RCA products can be tailor-designed by manipulating the circular template, RCA has been employed to generate complex DNA nanostructures such as DNA origami, nanotubes, nanoribbons and DNA based metamaterials. These functional RCA based nanotechnologies have been utilized for biodetection, drug delivery, designing bioelectronic circuits and bioseparation. In this review, we introduce the fundamental engineering principles used to design RCA nanotechnologies, discuss recently developed RCA-based diagnostics and bioanalytical tools, and summarize the use of RCA to construct multivalent molecular scaffolds and nanostructures for applications in biology, diagnostics and therapeutics.

Synthetic DNA motors have great potential to mimic natural protein motors in cells but the operation of synthetic DNA motors in living cells remains challenging and has not been demonstrated. Here we report a DNAzyme motor that operates in living cells in response to a specific intracellular target. The whole motor system is constructed on a 20 nm gold nanoparticle (AuNP) decorated with hundreds of substrate strands serving as DNA tracks and dozens of DNAzyme molecules each silenced by a locking strand. Intracellular interaction of a target molecule with the motor system initiates the autonomous walking of the motor on the AuNP. An example DNAzyme motor responsive to a specific microRNA enables amplified detection of the specific microRNA in individual cancer cells. Activated by specific intracellular targets, these self-powered DNAzyme motors will have diverse applications in the control and modulation of biological functions.

The Human Disease Ontology (DO) (http://www.disease-ontology.org), database has undergone significant expansion in the past three years. The DO disease classification includes specific formal semantic rules to express meaningful disease models and has expanded from a single asserted classification to include multiple-inferred mechanistic disease classifications, thus providing novel perspectives on related diseases. Expansion of disease terms, alternative anatomy, cell type and genetic disease classifications and workflow automation highlight the updates for the DO since 2015. The enhanced breadth and depth of the DO’s knowledgebase has expanded the DO’s utility for exploring the multi-etiology of human disease, thus improving the capture and communication of health-related data across biomedical databases, bioinformatics tools, genomic and cancer resources and demonstrated by a 6.6× growth in DO’s user community since 2015. The DO’s continual integration of human disease knowledge, evidenced by the more than 200 SVN/GitHub releases/revisions, since previously reported in our DO 2015 NAR paper, includes the addition of 2650 new disease terms, a 30% increase of textual definitions, and an expanding suite of disease classification hierarchies constructed through defined logical axioms.

Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.

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We present here a binding-induced fluorescence turn-on assay for protein detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (Ag NCs) using guanine-rich DNA sequences. In an example of an assay for human α-thrombin, two aptamers (Apt15 and Apt29) were used and were modified by including additional sequence elements. A 12-nucleotide (nt) sequence was used to link the first aptamer with a nanocluster nucleation sequence at the 5'-end. The second aptamer was linked through a complementary sequence (12-nt) to a G-rich overhang at the 3'-end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer and thereby bring the end of the G-rich overhang to close proximity to Ag NCs, resulting in a significant fluorescence enhancement. With this approach, a detection limit of 1 nM and a linear dynamic range of 5 nM-2 μM were achieved for human α-thrombin. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogeneous assay applications provided that two appropriate probes are available to bind with the same target molecule.

Dynamic DNA assemblies, including catalytic DNA circuits, DNA nanomachines, molecular translators, and reconfigurable nanostructures, have shown promising potential to regulate cell functions, deliver therapeutic reagents, and amplify detection signals for molecular diagnostics and imaging. However, such applications of dynamic DNA assembly systems have been limited to nucleic acids and a few small molecules, due to the limited approaches to trigger the DNA assemblies. Herein, we describe a binding-induced DNA strand displacement strategy that can convert protein binding to the release of a predesigned output DNA at room temperature with high conversion efficiency and low background. This strategy allows us to construct dynamic DNA assembly systems that are able to respond to specific protein binding, opening an opportunity to initiate dynamic DNA assembly by proteins.

An electrochemical DNAzyme sensor for sensitive and selective detection of lead ion (Pb(2+)) has been developed, taking advantage of catalytic reactions of a DNAzyme upon its binding to Pb(2+) and the use of DNA-Au bio-bar codes to achieve signal enhancement. A specific DNAzyme for Pb(2+) is immobilized onto an Au electrode surface via a thiol-Au interaction. The DNAzyme hybridizes to a specially designed complementary substrate strand that has an overhang, which in turn hybridizes to the DNA-Au bio-bar code (short oligonucleotides attached to 13 nm gold nanoparticles). A redox mediator, Ru(NH3)6(3+), which can bind to the anionic phosphate of DNA through electrostatic interactions, serves as the electrochemical signal transducer. Upon binding of Pb(2+) to the DNAzyme, the DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal of the substrate strand along with the DNA bio-bar code and the bound Ru(NH3)6(3+) from the Au electrode surface. The release of Ru(NH3)6(3+) results in lower electrochemical signal of Ru(NH3)6(3+) confined on the electrode surface. Differential pulse voltammetry (DPV) signals of Ru(NH3)6(3+) provides quantitative measures of the concentrations of Pb(2+), with a linear calibration ranging from 5 nM to 0.1 microM. Because each nanoparticle carries a large number of DNA strands that bind to the signal transducer molecule Ru(NH3)6(3+), the use of DNA-Au bio-bar codes enhances the detection sensitivity by five times, enabling the detection of Pb(2+) at a very low level (1 nM). The DPV signal response of the DNAzyme sensor is negligible for other divalent metal ions, indicating that the sensor is highly selective for Pb(2+). Although this DNAzyme sensor is demonstrated for the detection of Pb(2+), it has the potential to serve as a general platform for design sensors for other small molecules and heavy metal ions.