DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Transcriptional Inhibition of MicroRNA miR-122 by Small Molecules Reduces Hepatitis C Virus Replication in Liver Cells.

MicroRNAs (miRNAs) are noncoding RNA molecules of 22-24 nucleotides that are estimated to regulate thousands of genes in humans, and their dysregulation has been implicated in many diseases. MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and has been linked to the development of hepatocellular carcinoma and hepatitis C virus (HCV) infection. Its role in these diseases renders miR-122 a potential target for small-molecule therapeutics. Here, we report the discovery of a new sulfonamide class of small-molecule miR-122 inhibitors from a high-throughput screen using a luciferase-based reporter assay. Structure-activity relationship (SAR) studies and secondary assays led to the development of potent and selective miR-122 inhibitors. Preliminary mechanism-of-action studies suggest a role in the promoter-specific transcriptional inhibition of miR-122 expression through direct binding to the liver-enriched transcription factor hepatocyte nuclear factor 4α. Importantly, the developed inhibitors significantly reduce HCV replication in human liver cells.

DNA Computing: NOT Logic Gates See the Light.

DNA-based Boolean logic gates (for example, AND, OR, and NOT) can be assembled into complex computational circuits that generate an output signal in response to specific patterns of oligonucleotide inputs. However, the fundamental nature of NOT gates, which convert the absence of an input into an output, makes their implementation within DNA-based circuits difficult. Premature execution of a NOT gate before completion of its upstream computation introduces an irreversible error into the circuit. By utilizing photocaging groups, we developed a novel DNA gate design that prevents gate function until irradiation at a certain time point. Optical activation provides temporal control over circuit performance by preventing premature computation and is orthogonal to all other components of DNA computation devices. Using this approach, we designed NAND and NOR logic gates that respond to synthetic microRNA sequences. We further demonstrate the utility of the NOT gate within multilayer circuits in response to a specific pattern of four microRNAs.

High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning.

High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

High affinity nucleic acid analogues such as gammaPNA (γPNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a γPNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the γPNA probe, leaving an additional five γPNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity.

Small molecule inhibition of microRNA-21 expression reduces cell viability and microtumor formation.

MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of a large number of protein-coding genes and are implicated in a variety of biological processes such as development, differentiation, proliferation, and cell survival. Dysregulation of miRNAs has been attributed to the onset and progression of various human diseases, including cancer. MicroRNA-21 (miR-21), one of the most established oncogenic miRNAs, is found to be upregulated in a wide range of cancers making it an attractive therapeutic target. Employment of a luciferase-based live-cell reporter assay in a high-throughput screen of >300,000 small molecules led to the discovery of a new class of ether-amide miR-21 inhibitors. Following a structure-activity relationship study, an optimized lead molecule was found to inhibit miR-21 transcription. Furthermore, the inhibitor demonstrated cytotoxicity in a cervical cancer cell line via induction of apoptosis and was capable of reducing microtumor formation in a long-term clonogenic assay. Altogether, this work reports the discovery of a new small molecule inhibitor of miR-21 and demonstrates its potential as an alternative approach in cancer therapy.