DNA multi-bit non-volatile memory and bit-shifting operations using addressable electrode arrays and electric field-induced hybridization.

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations

Dynamic allosteric control of noncovalent DNA catalysis reactions

Allosteric modulation of catalysis kinetics is prevalent in proteins and has been rationally designed for ribozymes. Here, we present an allosteric DNA molecule that, in its active configuration, catalyzes a noncovalent DNA reaction. The catalytic activity is designed to be modulated by the relative concentrations of two DNA regulator molecules, one an inhibitor and the other an activator. Dynamic control of the catalysis rate is experimentally demonstrated via three cycles of up and down regulation by a factor of over 10. Unlike previous works, both the allosteric receptor and catalytic core are designed, rather than evolved. This allows flexibility in the sequence design and modularity in synthetic network construction

DNA-based communication in populations of synthetic protocells

Developing molecular communication platforms based on orthogonal communication channels is a crucial step towards engineering artificial multicellular systems. Here, we present a general and scalable platform entitled ‘biomolecular implementation of protocellular communication’ (BIO-PC) to engineer distributed multichannel molecular communication between populations of non-lipid semipermeable microcapsules. Our method leverages the modularity and scalability of enzyme-free DNA strand-displacement circuits to develop protocellular consortia that can sense, process and respond to DNA-based messages. We engineer a rich variety of biochemical communication devices capable of cascaded amplification, bidirectional communication and distributed computational operations. Encapsulating DNA strand-displacement circuits further allows their use in concentrated serum where non-compartmentalized DNA circuits cannot operate. BIO-PC enables reliable execution of distributed DNA-based molecular programs in biologically relevant environments and opens new directions in DNA computing and minimal cell technology

Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.Ministerio de Ciencia, Innovación y Universidades [BIO2016–75235-P

Split Aptameric Turn-On Fluorescence Sensor for Detection of Sequence Specific Nucleic Acid at Ambient Temperature

Nucleic acid amplification tests (NAATs) enable sensitive detection of low density infections that microscopy and rapid diagnostic test are not capable of detecting. They enable quantitative and qualitative nucleic acid detection, genotype analysis, and single nucleotide polymorphism (SNP) detection. Current state of the art molecular probes used with NAATs includes molecular beacon (MB), Taqman and its variations. This work presents novel molecular probe designed from Spinach and Dapoxyl aptamers. The aptamers are split into two parts (split aptamer), allowing greater sensitivity and selectivity towards fully complementary nucleic acid analyte. The major advantage of split aptamer probe over state-of-the-art fluorescent probes is its high selectivity: in the presence of a single base mismatched analyte, it produces only background fluorescence, even at room temperature. SSA is a promising tool for label-free analysis of nucleic acids at ambient temperatures. Split spinach aptamer (SSA) probes and split dapoxyl aptamer (SDA) for fluorescent analysis of nucleic acids were designed and tested. In both split aptamer design, two RNA or RNA/DNA or DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for fluorescent dye, which was accompanied by up to 270-fold and 69-fold increase in fluorescence. SSAr consisted entirely of ribonucleotides which potentially be expressed in live cells and used for imaging of specific mRNAs. For in vitro RNA/DNA analysis, SDA consisting of entirely DNA are preferable due to greater chemical stability, lower synthetic cost and reduced ability to form intramolecular structures. Additionally, we designed two DNA strands that function as an adapter for SSA and demonstrated how a single universal spinach aptamer (USSA) probe can be used to detect multiple (potentially any) nucleic acid sequences. USSA can be used for cost-efficient and highly selective analysis of even folded DNA and RNA analytes, as well as for the readout of outputs of DNA logic circuits

Molecular beacon strategies for sensing purpose

The improvement of nucleic acid probes as vital molecular engineering devices will cause a noteworthy contribution to developments in bioimaging, biosensing, and disorders diagnosis. The molecular beacon (MB) which was designed by Tyagi and Kramer in 1996, are loop-stem hairpin-designed oligonucleotides armed with a quencher and a dye (also named reporter groups) at the 30 or 50 ends. This construction allows that MBs in the absence of their target complementary molecules do not fluoresce. Through hybridization with their specific targets a spontaneous configuration change on MBs occur and the dye and quencher separate from each other, resulting in emitting the fluorescence. MBs are effective probes for biosensing because of their extraordinary target-specificity, unique structure, inherent fluorescent signal transduction mechanism, low background fluorescence emission, recognition without separation, and favorable thermodynamic properties. In comparison to other probes (such as linear DNA sequences), MBs with the same number of complementary nucleotides matching their target, are multitasking probes. They have advantages of thermodynamic and photostability, flexible ability for conjugation, higher efficient intrinsic signal switching, and ultra-sensitivity. MBs not only are useful for identifying a nucleic acid target but can also be employed for recognition of various non-nucleic acid goals, including heavy metals and cations, enzymes, cells, ATP, etc. Hence, this review highlights the potential of MBs in the improvement of biosensors and their usage in detection of different analytes such as miRNA, mRNA, cocaine, methamphetamine, actin, thrombin, heavy metal and cations and so on. (C) 2020 Elsevier B.V. All rights reserved.Peer reviewe

Nucleic acid i-motif structures in analytical chemistry

Under the appropriate experimental conditions of pH and temperature, cytosine-rich segments in DNA or RNA sequences may produce a characteristic folded structure known as an i-motif. Besides its potential role in vivo, which is still under investigation, this structure has attracted increasing interest in other fields due to its sharp, fast and reversible pH-driven conformational changes. This 'on/off' switch at molecular level is being used in nanotechnology and analytical chemistry to develop nanomachines and sensors, respectively. This paper presents a review of the latest applications of this structure in the field of chemical analysis