Harnessing DNA computing and nanopore decoding for practical applications: from informatics to microRNA-targeting diagnostics.

DNA computing represents a subfield of molecular computing with the potential to become a significant area of next-generation computation due to the high programmability inherent in the sequence-dependent molecular behaviour of DNA. Recent studies in DNA computing have extended from mathematical informatics to biomedical applications, with a particular focus on diagnostics that exploit the biocompatibility of DNA molecules. The output of DNA computing devices is encoded in nucleic acid molecules, which must then be decoded into human-recognizable signals for practical applications.

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell.

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging.

DNA circuit-based immunoassay for ultrasensitive protein pattern classification.

Cytokines are important immune modulators, and pivotal biomarkers for the diagnostic of various diseases. In standard analytical procedure, each protein is detected individually, using for instance gold standard ELISA protocols or nucleic acid amplification-based immunoassays. In recent years, DNA nanotechnology has been employed for creating sophisticated biomolecular systems that perform neuromorphic computing on molecular inputs, opening the door to concentration pattern recognition for biomedical applications.

Time-encoded electrical detection of trace RNA biomarker by integrating programmable molecular amplifier on chip.

One of the serious challenges facing modern point-of-care (PoC) molecular diagnostic platforms relate to reliable detection of low concentration biomarkers such as nucleic acids or proteins in biological samples. Non-specific analyte-receptor interactions due to competitive binding in the presence of abundant molecules, inefficient mass transport and very low number of analyte molecules in sample volume, in general pose critical hurdles for successful implementation of such PoC platforms for clinical use. Focusing on these specific challenges, this work reports a unique PoC biosensor that combines the advantages of nanoscale biologically-sensitive field-effect transistor arrays (BioFET-arrays) realized in a wafer-scale top-down nanofabrication as high sensitivity electrical transducers with that of sophisticated molecular programs (MPs) customized for selective recognition of analyte miRNAs and amplification resulting in an overall augmentation of signal transduction strategy.

Functional analysis of single enzymes combining programmable molecular circuits with droplet-based microfluidics.

The analysis of proteins at the single-molecule level reveals heterogeneous behaviours that are masked in ensemble-averaged techniques. The digital quantification of enzymes traditionally involves the observation and counting of single molecules partitioned into microcompartments via the conversion of a profluorescent substrate. This strategy, based on linear signal amplification, is limited to a few enzymes with sufficiently high turnover rate.

Enzyme Self-Selection Using Molecular Programs.

Directed evolution provides a powerful route for enzyme engineering. State-of-the-art techniques functionally screen up to millions of enzyme variants using high throughput microfluidic sorters, whose operation remains technically challenging. Alternatively, self-selection methods, analogous to complementation strategies, open the way to even higher throughputs, but have been demonstrated only for a few specific activities.

Silicon chambers for enhanced incubation and imaging of microfluidic droplets.

Droplet microfluidics has become a powerful tool in life sciences, underlying digital assays, single-cell sequencing or directed evolution, and it is making foray in physical sciences as well. Imaging and incubation of droplets are crucial, yet they are encumbered by the poor optical, thermal and mechanical properties of PDMS, a material commonly used in microfluidics labs. Here we show that Si is an ideal material for droplet chambers.

Molecular Computation for Molecular Classification.

DNA as an informational polymer has, for the past 30 years, progressively become an essential molecule to rationally build chemical reaction networks endowed with powerful signal-processing capabilities. Whether influenced by the silicon world or inspired by natural computation, molecular programming has gained attention for diagnosis applications. Of particular interest for this review, molecular classifiers have shown promising results for disease pattern recognition and sample classification.

Programmable Ultrasensitive Molecular Amplifier for Digital and Multiplex MicroRNA Quantification.

Digital bioassays, popularized by digital PCR, provide some of the most robust and accurate methods for nucleic acid quantification. In this chapter, we detail a protocol for digital, isothermal, and multiplex detection of microRNAs, which relies on a recently developed amplification method. Our approach uses programmable ultrasensitive molecular amplifiers (PUMAs) to reveal the presence of target microRNAs randomly isolated in picoliter-size microfluidic droplets.

Accurate gene consensus at low nanopore coverage.

Background: Nanopore technologies allow high-throughput sequencing of long strands of DNA at the cost of a relatively large error rate. This limits its use in the reading of amplicon libraries in which there are only a few mutations per variant and therefore they are easily confused with the sequencing noise. Consensus calling strategies reduce the error but sacrifice part of the throughput on reading typically 30 to 100 times each member of the library.

Lessons from Biomass Valorization for Improving Plastic-Recycling Enzymes.

Synthetic polymers such as plastics exhibit numerous advantageous properties that have made them essential components of our daily lives, with plastic production doubling every 15 years. The relatively low cost of petroleum-based polymers encourages their single use and overconsumption. Synthetic plastics are recalcitrant to biodegradation, and mismanagement of plastic waste leads to their accumulation in the ecosystem, resulting in a disastrous environmental footprint.

A small-molecule chemical interface for molecular programs.

In vitro molecular circuits, based on DNA-programmable chemistries, can perform an increasing range of high-level functions, such as molecular level computation, image or chemical pattern recognition and pattern generation. Most reported demonstrations, however, can only accept nucleic acids as input signals. Real-world applications of these programmable chemistries critically depend on strategies to interface them with a variety of non-DNA inputs, in particular small biologically relevant chemicals.

Advances in multiplexed techniques for the detection and quantification of microRNAs.

MicroRNA detection is currently a crucial analytical chemistry challenge: almost 2000 papers were referenced in PubMed in 2018 and 2019 for the keywords "miRNA detection method". MicroRNAs are potential biomarkers for multiple diseases including cancers, neurodegenerative and cardiovascular diseases. Since miRNAs are stably released in bodily fluids, they are of prime interest for the development of non-invasive diagnosis methods, such as liquid biopsies.

Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits.

Ubiquitous post-transcriptional regulators in eukaryotes, microRNAs are currently emerging as promising biomarkers of physiological and pathological processes. Multiplex and digital detection of microRNAs represents a major challenge toward the use of microRNA signatures in clinical settings. The classical reverse transcription polymerase chain reaction quantification approach has important limitations because of the need for thermocycling and a reverse transcription step.

Quantifying the Performance of Micro-Compartmentalized Directed Evolution Protocols.

High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants.

Isothermal digital detection of microRNAs using background-free molecular circuit.

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout.

Streamlined digital bioassays with a 3D printed sample changer.

Droplet-based microfluidics has permeated many areas of life sciences including biochemistry, biology and medicine. Water-in-oil droplets act as independent femto- to nano-liter reservoirs, enabling the parallelization of (bio)chemical reactions with a minimum sample input. Among the range of applications spanned by droplet microfluidics, digital detection of biomolecules, using Poissonian isolation of single molecules in compartments, has gained considerable attention due to the high accuracy, sensitivity and robustness of these methods.

Emerging isothermal amplification technologies for microRNA biosensing: Applications to liquid biopsies.

The potential of microRNAs (miRNAs) as biomarker candidates in clinical practice for diagnosis, prognosis and treatment response prediction, especially in liquid biopsies, has led to a tremendous demand for techniques that can detect these molecules rapidly and accurately. Hence, numerous achievements have been reported recently in miRNA research. In this review, we discuss the challenges associated with the emerging field of miRNA detection, which are linked to the intrinsic properties of miRNAs, advantages and drawbacks of the currently available technologies and their potential applications in clinical research.

Selection strategies for randomly partitioned genetic replicators.

The amplification cycle of many replicators (natural or artificial) involves the usage of a host compartment, inside of which the replicator expresses phenotypic compounds necessary to carry out its genetic replication. For example, viruses infect cells, where they express their own proteins and replicate. In this process, the host cell boundary limits the diffusion of the viral protein products, thereby ensuring that phenotypic compounds, such as proteins, promote the replication of the genes that encoded them.

Spatiotemporal control of DNA-based chemical reaction network via electrochemical activation in microfluidics.

In recent years, DNA computing frameworks have been developed to create dynamical systems which can be used for information processing. These emerging synthetic biochemistry tools can be leveraged to gain a better understanding of fundamental biology but can also be implemented in biosensors and unconventional computing. Most of the efforts so far have focused on changing the topologies of DNA molecular networks or scaling them up.

Synthesis and materialization of a reaction-diffusion French flag pattern.

During embryo development, patterns of protein concentration appear in response to morphogen gradients. These patterns provide spatial and chemical information that directs the fate of the underlying cells. Here, we emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material.

Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.

Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template.

Boosting functionality of synthetic DNA circuits with tailored deactivation.

Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network.