DNA nanotechnology for nucleic acid analysis: multifunctional molecular DNA machine for RNA detection

Abstract

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...

Multiplex detection of extensively drug resistant tuberculosis using binary deoxyribozyme sensors

“…In recent studies, we have demonstrated innovative approaches to further improve the sensitivity of BiDz assays via multicomponent nucleic acid assemblies that “trap” substrate molecules near sites of BiDz assembly and unwind RNA analytes (Cox et al 2016a; Cox et al 2016b). We have also recently reported a proof-of-principle study for an alternative visual, colorimetric readout which would be ideal for point-of-care implementation (Gerasimova et al 2013).…”

A mutation-resistant deoxyribozyme OR gate for highly selective detection of viral nucleic acids

“…(A) Binary DZ (BiDZ) sensor for fluorescence detection of specific DNA or RNA analytes. 7 DNA strands DZa and DZb hybridize to a targeted analyte and form a DZ catalytic core, which cleaves a fluorophore (F) and a quencher (Q) labelled F-sub followed by separation of F from Q associated with an increased fluorescence. (B) Three EV71-specific fragments (targets I, II and III) were chosen to be targeted by the DZ OR gate, which consisted of 3 series of BiDZ sensors: DZ-I, DZ-II and DZ-III.…”

“…1A). 7 BiDZ consists of two DNA strands DZa and DZb, which hybridize to a targeted analyte and form a catalytic core of RNA-cleaving DZ. The DZ cleaves a fluorophore and a quencher labelled fluorogenic substrate (F-sub, Table S3, ESI † ), followed by an increase in fluorescence.…”

“…Indeed, the two-component design enables high selectivity even at ambient temperatures. 7 The selectivity is improved due to the catalytic signal amplification: one activated DZ core can cleave multiple F-sub molecules, thus increasing the output signal over time.…”

Nucleic Acid Analysis Using Multifunctional Hybridization Sensors

“…Each MHP component serves a specific function, thus enabling simultaneous improvement of multiple key characteristics. My presentation will cover the design of a probe that can differentiate single nucleotide substitutions in DNA in the entire temperature interval of 5-40 °C [1]; molecular machine that tightly binds the RNA analyte while remaining highly selective [2]; a strategy for the recognition of highly variable viral genomes with high selectivity [3].…”