The deamination of adenosine to inosine is an important modification in nucleic acids that functionally recodes the identity of the nucleobase to a guanosine. Current methods to analyze and detect this single nucleotide change, such as sequencing and PCR, typically require time-consuming or costly procedures. Alternatively, fluorescent "turn-on"probes that result in signal enhancement in the presence of target are useful tools for real-time detection and monitoring of nucleic acid modification. Here we describe forced-intercalation PNA (FIT-PNA) probes that are designed to bind to inosine-containing nucleic acids and use thiazole orange (TO), 4-dimethylamino-naphthalimide (4DMN), and malachite green (MG) fluorogenic dyes to detect A-to-I editing events. We show that incorporation of the dye as a surrogate base negatively affects the duplex stability but does not abolish binding to targets. We then determined that the identity of the adjacent nucleobase and temperature affect the overall signal and fluorescence enhancement in the presence of inosine, achieving an 11-fold increase, with a limit of detection (LOD) of 30 pM. We determine that TO and 4DMN probes are viable candidates to enable selective inosine detection for biological applications.

Adenosine to-inosine (A-to-I) RNA editing is a conserved post-transcriptional modification that is critical for a variety of cellular processes. A-to-I editing is widespread in nearly all types of RNA, directly imparting significant global changes in cellular function and behavior. Dysfunctional RNA editing is also implicated in a number of diseases, and A-to-I editing activity is rapidly becoming an important biomarker for early detection of cancer, immune disorders, and neurodegeneration. While millions of sites have been identified, the biological function of the majority of these sites is unknown, and the regulatory mechanisms for controlling editing activity at individual sites is not well understood. Robust detection and mapping of A-to-I editing activity throughout the transcriptome is vital for understanding these properties and how editing affects cellular behavior. However, accurately identifying A-to-I editing sites is challenging because of inherent sampling errors present in RNA-seq. We recently developed Endonuclease V immunoprecipitation enrichment sequencing (EndoVIPER-seq) to directly address this challenge by enrichment of A-to-I edited RNAs prior to sequencing. This protocol outlines how to process cellular RNA, enrich for A-to-I edited transcripts with EndoVIPER pulldown, and prepare libraries suitable for generating RNA-seq data. © 2020 Wiley Periodicals LLC. Basic Protocol 1: mRNA fragmentation and glyoxalation Basic Protocol 2: EndoVIPER pulldown Basic Protocol 3: RNA-seq library preparation and data analysis.

Fluorophore bioconjugation to proteins, nucleic acids, and other important molecules can provide a powerful approach to sensing, imaging, and quantifying chemical and biological processes. One of the most prevalent methods for fluorophore attachment is through the formation of amide bonds, which are often facilitated by coupling agents to activate carboxylic acid moieties for subsequent nucleophilic attack by amines. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) is among the most popular of these coupling agents for bioconjugation due to its ability to facilitate amide bond formation in water. After observing quenching of 5-fluoresceinamine (5-FAM)-conjugated oligonucleotides in the presence of DMTMM, we sought to evaluate the magnitude and scope of this challenge by surveying the effect of DMTMM on a range of fluorescent dyes. A higher quenching effect was consistently observed for xanthene dyes compared to that for cyanine dyes. Further analysis of the impact of DMTMM on FAM shows that quenching occurs independently of whether the dye is free in solution or attached to an oligonucleotide or antibody. Furthermore, we found that FAM-conjugated DNA was unable to recover its fluorescence after the removal of DMTMM, and UV-vis and NMR analyses suggest the formation of new products, such as an adduct formed between FAM and the dimethoxytriazine of DMTMM. As such, DMTMM at high concentrations is not recommended for coupling reactions where targets are fluorescently labeled. This research serves as a word of caution to those utilizing xanthene-containing fluorophores in bioconjugation reactions involving DMTMM.

Small molecule contaminants pose a significant threat to the environment and human health. While regulations are in place for allowed limits in many countries, detection and remediation of contaminants in more resource-limited settings and everyday environmental sources remains a challenge. Functional nucleic acids, including aptamers and DNA enzymes, have emerged as powerful options for addressing this challenge due to their ability to non-covalently interact with small molecule targets. The goal of this perspective is to outline recent efforts toward the selection of aptamers for small molecules and describe their subsequent implementation for environmental applications. Finally, we provide an outlook that addresses barriers that hinder these technologies from being widely adopted in field friendly settings and propose a path forward toward addressing these challenges.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications. This journal is

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is “click”-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.

Creating accurate maps of A-to-I RNA editing activity is vital to improving our understanding of the biological role of this process and harnessing it as a signal for disease diagnosis. Current RNA sequencing techniques are susceptible to random sampling limitations due to the complexity of the transcriptome and require large amounts of RNA material, specialized instrumentation, and high read counts to accurately interrogate A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA. While Mg2+ enables eEndoV to catalyze RNA cleavage, we show that similar levels of Ca2+ instead promote binding of inosine without cleavage and thus enable high affinity capture of inosine in RNA. We leverage this capability to demonstrate EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) as a facile and effective method to enrich A-to-I edited transcripts prior to RNA-seq, producing significant increases in the coverage and detection of identified editing sites. We envision the use of this approach as a straightforward and cost-effective strategy to improve the epitranscriptomic informational density of RNA samples, facilitating a deeper understanding of the functional roles of A-to-I editing. ©

Adenosine-to-inosine (A-to-I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. Here, we utilize Endonuclease V (EndoV), which binds specifically to inosine in RNA, to develop an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay with chemically synthesized oligonucleotides. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development.

Branched DNAs (bDNAs) having comb-like structures have found wide utility in molecular diagnostics and DNA nanotechnology. bDNAs can be generated either by designing and assembling linear DNA molecules into rigid non-covalent structures or by using an orthogonally protected branching unit to synthesize covalently linked structures. Despite the advantages of the covalently linked structures, use of this motif has been hampered by the challenging synthesis of appropriately protected branching monomers. We report the facile synthesis of a branching monomer having orthogonal DMT and Lev protecting groups using readily available δ-velarolactone and 1,3-diaminopropan-2-ol. Using this branching monomer, a comb-shaped bDNA was synthesized having three different DNA arms. The synthesis and hybridization capability of the bDNA was assessed by fluorescence microscopy using fluorescently labeled complementary and mismatched DNA probes. Convenient access to an orthogonally protected branching monomer is anticipated to accelerate applications of bDNAs in applications including diagnostics, biosensing, gene-profiling, DNA computing, multicolor imaging, and nanotechnology.