The Nobel prize in chemistry in 2016 was awarded for 'the design and synthesis of molecular machines'. Here we designed and assembled a molecular machine for the detection of specific RNA molecules. An association of several DNA strands, named multifunctional DNA machine for RNA analysis (MDMR1), was designed to (i) unwind RNA with the help of RNA-binding arms, (ii) selectively recognize a targeted RNA fragment, (iii) attract a signal-producing substrate and (iv) amplify the fluorescent signal by catalysis. MDMR1 enabled detection of 16S rRNA at concentrations ~24 times lower than that by a traditional deoxyribozyme probe.Hybridization probes have been used for the detection of specific DNA and RNA sequences for the last 55 years. 1a Common challenges of hybridization probes include insufficient sensitivity and selectivity. 1 Detection of specific RNA molecules is particularly challenging due to the stable secondary structures that inhibit or even prevent their interactions with hybridization probes. 2 For example, some molecular beacon probes, fluorophore-and quencher-labelled DNA stem-loop structures, 3 were previously shown to fail in detecting folded RNA and DNA molecules. 4 Probes based on nucleic acid analogues, e.g. peptide nucleic acids and locked nucleic acids, are required to tightly bind and unwind structured RNAs. 5 Here we took advantage of recent developments of DNA nanotechnology 6 to design a multifunctional platform that enables (i) unfolding of a specific RNA analyte, (ii) specific recognition of a targeted fragment, (iii) enhanced delivery of a signal-producing substrate to a target-activated catalytic sensor, and (iv) signal amplification by catalysis. We named the platform 'multifunctional DNA machine 7 for RNA analysis of the 1st generation' (MDMR1).MDMR1 was designed based on binary deoxyribozyme (BiDZ) probe, 8 in which two unmodified DNA strands, DZ a and DZ b , hybridize to the abutting positions of a complementary DNA or RNA analyte to form the catalytic core of an RNA-cleaving deoxyribozyme (Fig. 1A). 9 The active core cleaves a fluorophore-and a quencher-labelled F_sub strand, which results in a production of a fluorescent signal. For BiDZ, as the signal † Electronic supplementary information (ESI) available: Detailed experimental procedure and the structure of MDMR1; data used for calculations of LODs, as well as kinetics of MDRRA1 that lacks RNA-binding arm or Hook strand. See DOI: 10.1039/c6cc06889hCorrespondence to: D. M. Kolpashchikov. 1,3b However, the LOD of BiDZ sensors is compromised when long and highly folded RNA is to be analysed. In this case, the access of the BiDZ probe to the targeted analyte fragment is limited resulting in ~10-25 fold decrease in LOD in comparison to an unstructured analyte. 8h,l We therefore seek to equip the BiDZ probe with the RNAunwinding function. We reasoned that the addition of an RNA-unwinding function to the BiDZ probe will help to facilitate its access to the targeted fragment and achieve lower LODs. Furthermore, deoxyriboz...

Some natural enzymes increase the rate of diffusion-limited reactions by facilitating substrate flow to their active sites. Inspired by this natural phenomenon, we developed a strategy for efficient substrate delivery to a deoxyribozyme (DZ) catalytic sensor. This resulted in 3- to 4-fold increase in its sensitivity and up to 9-fold improvement of the detection limit. The reported strategy can be used to enhance catalytic efficiency of diffusion-limited enzymes and to improve sensitivity of enzyme-based biosensors.

Current diagnostic tools for Mycobacterium tuberculosis (Mtb) have many disadvantages including low sensitivity, slow turnaround times, or high cost. Accurate, easy to use, and inexpensive point of care molecular diagnostic tests are urgently needed for the analysis of multidrug resistant (MDR) and extensively drug resistant (XDR) Mtb strains that emerge globally as a public health threat. In this study, we established proof-of-concept for a novel diagnostic platform (TB-DzT) for Mtb detection and the identification of drug resistant mutants using binary deoxyribozyme sensors (BiDz). TB-DzT combines a multiplex PCR with single nucleotide polymorphism (SNP) detection using highly selective BiDz sensors targeting loci associated with species typing and resistance to rifampin, isoniazid and fluoroquinolone antibiotics. Using the TB-DzT assay, we demonstrated accurate detection of Mtb and 5 mutations associated with resistance to three anti-TB drugs in clinical isolates. The assay also enables detection of a minority population of drug resistant Mtb, a clinically relevant scenario referred to as heteroresistance. Additionally, we show that TB-DzT can detect the presence of unknown mutations at target loci using combinatorial BiDz sensors. This diagnostic platform provides the foundation for the development of cost-effective, accurate and sensitive alternatives for molecular diagnostics of MDR- and XDR-TB.

Differential receptors use an array of sensors to recognize analytes. Each sensor in the array can recognize not one, but several analytes with different rates, so a single analyte triggers a response of several sensors in the array. The receptor thus produces a pattern of signals that is unique for each analyte, thereby enabling identification of a specific analyte by producing a “fingerprint” pattern. We applied this approach for the analysis of DNA sequences of Mycobacterium tuberculosis strains that differ by single nucleotide substitutions in the 81‐bp hot‐spot region that imparts rifampin resistance. The technology takes advantage of the new multicomponent, selfassembling sensor, which produces a fluorescent signal in the presence of specific DNA sequences. A differential fluorescent receptor (DFR) contained an array of three such sensors and differentiated at least eight DNA sequences. The approach requires only one molecular‐beacon‐like fluorescent reporter, which can be used by all three sensors. The DFR developed in this study represents a cost‐efficient alternative to molecular diagnostic technologies that use fluorescent hybridization probes.