Complementary RNA Strands Research Articles

Carrier capability of halloysite nanotubes for the intracellular delivery of antisense PNA targeting mRNA of neuroglobin gene

Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently.Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.

Exploring the DNA2-PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups’ research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.

A Convenient Method for the Synthesis of 2′‐O‐Cyanoethylated Nucleotides and Their Application in the Solid‐Phase Synthesis of Related RNA Analogs

AbstractAlthough small interfering RNA (siRNA) is a key player among gene inhibition therapeutics, there are many obstacles to the development of siRNA drugs due to inherent properties of oligonucleotides, including the unsatisfactory stability of unmodified siRNA, poor pharmacokinetic distribution, and the toxicity induced by off‐target effects. To maximize treatment potency, chemical modification of siRNA has undoubtedly been the most successful strategy by far. Widely applied modifications include phosphorothioate linkages, 2′‐O‐methyl modifications, and 2′‐fluoro modifications, among others. To extend the family of chemical modifications for oligonucleotides, 2′‐O‐cyanoethylated RNA analogs were developed through the replacement of the 2′‐hydroxyl group with a 2′‐O‐cyanoethyl group (‐OCH2CH2CN). This modification can provide several advantages over unmodified RNA, such as increased stability, improved binding affinity to complementary DNA or RNA strands, and resistance to degradation by cellular nucleases. The 2′‐O‐cyanoethyl‐modified RNAs not only are applied in RNA silencing machinery but also act as research tools for studying RNA structure and function or for developing RNA‐based diagnostics. Therefore, the efficient synthesis, deprotection, purification, and characterization of 2′‐O‐cyanoethylated RNAs deserves more attention. This protocol describes the chemical synthesis of 2′‐O‐cyanoethylated nucleotides and the solid‐phase synthesis, deprotection, and purification of 2′‐O‐cyanoethylated RNAs. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Preparation of 6‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl adenosine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 2: Preparation of 4‐N‐acetyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl cytidine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 3: Preparation of 2‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl guanine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 4: Preparation of 5′‐O‐dimethoxytrityl‐2′‐O‐2‐cyanoethyl uridine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 5: Solid‐phase synthesis of 2′‐O‐cyanoethylated RNA analogsBasic Protocol 6: Deprotection and purification of synthesized 2′‐O‐cyanoethyl‐RNAs

New Oligonucleotide 2'-O-Alkyl N3'→P5' (Thio)-Phosphoramidates as Potent Antisense Agents: Physicochemical Properties and Biological Activity.

We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'→P5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.

Synthesis and properties of a novel modified nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid

2′-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2′-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2′-methylamino-LNA (ALNA[Me]), which is a classical 2′-amino-LNA derivative, and also on 2′,4′-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics.

C5-pyrimidine-functionalized morpholino oligonucleotides exhibit differential binding affinity, target specificity and lipophilicity.

C5-substituted uridine and cytidine morpholino chlorophosphoramidate monomers were synthesized and incorporated into a 12-mer Phosphorodiamidate Morpholino Oligonucleotide (PMO) using semi-automated solid phase synthesis. PMOs with most of the tested pyrimidine C5-substitutions have significantly increased thermal stability when bound to the complementary RNA strand relative to the PMO. They exhibit higher binding with RNA than DNA. CD-spectra show B-type helical conformation of duplexes. HPLC analysis indicates their greater lipophilicity compared to regular PMOs. These chemical modifications have significant potential towards the development of better antisense technologies.

2′-N-Alkylaminocarbonyl-2′-amino-LNA: Synthesis, duplex stability, nuclease resistance, and in vitro anti-microRNA activity

2′-Amino-LNA has the potential to acquire various functions through chemical modification at the 2′-nitrogen atom. This study focused on 2′-N-alkylaminocarbonyl 2′-amino-LNA, which is a derivative of 2′-amino-LNA. We evaluated its practical usefulness as a chemical modification of anti-miRNA oligonucleotide. The synthesis of phosphoramidites of 2′-N-alkylaminocarbonyl substituted 2′-amino-LNA bearing thymine and 5-methylcytosine proceeded in good yields. Incorporating the 2′-N-alkylaminocarbonyl-2′-amino-LNA monomers into oligonucleotides improved the duplex stability for complementary RNA strands and robust nuclease resistance. Moreover, 2′-N-alkylaminocarbonyl-2′-amino-LNA is a promising scaffold that significantly increases the potency of anti-miRNA oligonucleotides.

ASCENDING AORTIC ANEURYSMS: SURGERY, RMODELLING & AI

ASCENDING AORTIC ANEURYSMS: SURGERY, RMODELLING & AI

Analysis of luciferase dsRNA production during baculovirus infection of Hi5 cells: RNA hairpins expressed by very late promoters do not trigger gene silencing.

The baculovirus expression vector system (BEVS) has become an important platform for the expression of recombinant proteins and is especially useful for the production of large protein complexes such as virus-like particles (VLPs). An important application for VLPs is their use as vehicles for targeted delivery of drugs or toxins which requires the development of methods for efficient loading with the intended cargo. Our research intends to employ the BEVS for the production of VLPs for the delivery of insecticidal dsRNA molecules to targeted insect pests (as "dsRNA-VLPs"). A convenient strategy would be the co-expression of long dsRNAs with viral capsid proteins and their simultaneous encapsulation during VLP assembly but the capacity of the BEVS for the production of long dsRNA has not been assessed so far. In this study, the efficiency of production of long RNA hairpins targeting the luciferase gene ("dsLuc") by the polyhedrin promoter during baculovirus infection was evaluated. However, RNAi reporter assays could not detect significant amounts of dsLuc in Hi5 cells infected with recombinant baculovirus, even in the presence of co-expressed dsRNA-binding protein B2-GFP or the employment of the MS2-MCP system. Nevertheless, dot blot analyses using anti-dsRNA antibody revealed that baculovirus-mediated expression of B2-GFP resulted in significant increases in dsRNA levels in infected cells that may correspond to hybridized complementary viral transcripts. Using B2-GFP as a genetically encoded sensor, dsRNA foci were detected in the nuclei that partially co-localized with DAPI staining, consistent with their localization at the virogenic stroma. Co-localization experiments with the baculovirus proteins vp39, Ac93, ODV-E25 and gp64 indicated limited overlap between B2-GFP and the ring zone compartment where assembly of nucleocapsids and virions occurs. Stability experiments showed that exogenous dsRNA is resistant to degradation in extracts of non-infected and infected Hi5 cells and it is proposed that strong unwinding activity at the virogenic stroma in the infected nuclei may neutralize the annealing of complementary RNA strands and block the production of long dsRNAs. Because the strong stability of exogenous dsRNA, transfection can be explored as an alternative method for delivery of cargo for dsRNA-VLPs during their assembly in baculovirus-infected Hi5 cells.

Sequence-Specific Features of Short Double-Strand, Blunt-End RNAs Have RIG-I- and Type 1 Interferon-Dependent or -Independent Anti-Viral Effects.

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.

Repurposing Molnupiravir for COVID-19: The Mechanisms of Antiviral Activity.

Molnupiravir is a β-d-N4-hydroxycytidine-5′-isopropyl ester (NHC) compound that exerts antiviral activity against various RNA viruses such as influenza, SARS, and Ebola viruses. Thus, the repurposing of Molnupiravir has gained significant attention for combatting infection with SARS-CoV-2, the etiological agent of COVID-19. Recently, Molnupiravir was granted authorization for the treatment of mild-to-moderate COVID-19 in adults. Findings from in vitro experiments, in vivo studies and clinical trials reveal that Molnupiravir is effective against SARS-CoV-2 by inducing viral RNA mutagenesis, thereby giving rise to mutated complementary RNA strands that generate non-functional viruses. To date, the data collectively suggest that Molnupiravir possesses promising antiviral activity as well as favorable prophylactic efficacy, attributed to its effective mutagenic property of disrupting viral replication. This review discusses the mechanisms of action of Molnupiravir and highlights its clinical utility by disabling SARS-CoV-2 replication, thereby ameliorating COVID-19 severity. Despite relatively few short-term adverse effects thus far, further detailed clinical studies and long-term pharmacovigilance are needed in view of its mutagenic effects.

A Strategy for Gene Knockdown in Dinoflagellates.

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Abstract 2162: Spatially resolved image-based transcriptomics using high-throughput single-molecule fluorescence in situ hybridization (HITSFISH)

Abstract Single-cell transcriptomics enables the study of heterogeneity by uncovering the transcriptome of individual cells, which is averaged out in bulk analyses. This facilitates the grouping of cells based on similar transcriptomic profiles, potentially identifying cell subpopulations, such as rare tumor cells that mediate drug resistance or undergo metastasis. Image-based transcriptomic techniques can go a step further in uniquely profiling cells, by providing information about the spatial co-ordinates of transcripts and cells, whilst shedding light on cell morphology for phenotypic characterization. Single-molecule Fluorescence in situ hybridization (smFISH) is one image-based method in which fluorescently labeled tiling-oligonucleotide (TO) probes are bound along the length of complementary RNA strands, allowing for the detection of transcripts at nanometer-scale resolution. However, smFISH data analysis has been difficult to implement at scale due to the tradeoff between transcript resolution and imaging area, as well as the lack of automated feature detection tools. To address this issue, we have developed a pipeline called HIgh-Throughput Single-molecule FISH (HITSFISH), which uniquely combines smFISH with structured-illumination microscopy allowing for large-area high-resolution imagery with automated feature detection and analysis. We have validated our platform by showing high transcriptomic concordance between HITSFISH and other standard methods such as bulk RNA-seq and qPCR, demonstrating its ability to accurately measure RNA levels. With our pipeline we look to answer questions pertaining to the spatiotemporal landscape of gene expression within and across normal and cancerous cells. In addition, by using immunofluorescence in tandem with our platform, we can study transcript localization at various subcellular landmarks, such as membraneless organelles, to gain insight on subcellular mechanisms of gene regulation. Citation Format: Andrej Coleski, Sethu Pitchiaya, Jeremy D'Silva, Marcin Cieslik, Arul Chinnaiyan. Spatially resolved image-based transcriptomics using high-throughput single-molecule fluorescence in situ hybridization (HITSFISH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2162.

From Prebiotic Chemistry to Supramolecular Biomedical Materials: Exploring the Properties of Self-Assembling Nucleobase-Containing Peptides.

Peptides and their synthetic analogs are a class of molecules with enormous relevance as therapeutics for their ability to interact with biomacromolecules like nucleic acids and proteins, potentially interfering with biological pathways often involved in the onset and progression of pathologies of high social impact. Nucleobase-bearing peptides (nucleopeptides) and pseudopeptides (PNAs) offer further interesting possibilities related to their nucleobase-decorated nature for diagnostic and therapeutic applications, thanks to their reported ability to target complementary DNA and RNA strands. In addition, these chimeric compounds are endowed with intriguing self-assembling properties, which are at the heart of their investigation as self-replicating materials in prebiotic chemistry, as well as their application as constituents of innovative drug delivery systems and, more generally, as novel nanomaterials to be employed in biomedicine. Herein we describe the properties of nucleopeptides, PNAs and related supramolecular systems, and summarize some of the most relevant applications of these systems.

Identification of Potential Key Agents for Targeting RNA-Dependent RNA Polymerase of SARS-CoV-2 by Integrated Analysis and Virtual Drug Screening.

BackgroundRNA-dependent RNA polymerase (RdRp) is the key enzyme responsible for the SARS-CoV-2 replication process and catalyzes the synthesis of complementary minus strand RNA and genomic plus strand RNA, often recognized as good targets for antiviral drugs.Materials and MethodsA systematic screening of existing antiviral compounds, family analysis, conserved domain analysis, three-dimensional structure modeling, drug virtual screening, and bioassays were performed to identify agents that potentially targeted RNA-dependent RNA polymerase of SARS-CoV-2.ResultsFour thousand nine hundred and forty seven antiviral lead compounds were selected and evaluated by systematic screening. Of these, 359 agents were screened by family analysis and conserved domain analysis. They were further analyzed by three-dimensional structure modeling, virtual drug screening, and bioassays. The results identified 102 agents with potential for repurposing to target the RNA-dependent RNA polymerase of SARS-CoV-2.ConclusionThis study identified 102 key agents with potential anti-SARS-CoV-2 RNA-dependent RNA polymerase function and prospects of rapid clinical application for the treatment of COVID-19.

A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates.

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage.

Peptide Nucleic Acid Conjugates of Quinone Methide Precursors Alkylate Ribonucleic Acid after Activation with Light.

Quinone methide precursors 2 and 3 were protected with a photoreactive 2-nitrobenzyl group and conjugated to peptide nucleic acids (PNA) using a Huisgen click reaction. After brief irradiation at 365 nm, cross-linking with complementary RNA strands started and was analyzed with an ALFexpress sequencer. When this method was used, the gel temperature had a major influence on apparent rates. Quinone methides are known to form transient as well as stable bonds with nucleotides. Although both were detected at 25 °C, analysis at 57 °C only recorded the stable types of cross-links, suggesting much slower alkylation kinetics. Linker 11 allowed us to attach quinone methides to internal positions of the PNA/RNA duplex and to capture a model of miR-20a with good efficiency.

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Parlez-vous protein?

If you’ve ever learned a language, says Jen Heemstra of Emory University, there are two levels of understanding. First you learn to read text and comprehend it. Then you begin speaking the language and interacting with others. In Heemstra’s lab, she says her team members are even more ambitious: the group is teaching molecules to speak both nucleic acid and protein languages simultaneously (J. Am. Chem. Soc. 2019, DOI: 10.1021/jacs.9b09146). (Heemstra partners with C&EN to write a monthly column, Office Hours.) Peptide nucleic acids (PNAs) are artificial polymers that were originally invented in 1991 to be synthetic mimics of DNA. Instead of having a deoxyribose phosphate backbone like DNA, PNA has a pseudopeptide polymer backbone to which nucleobases are linked. The resulting biopolymer is stabler than DNA, yet it can still “speak,” or interact, with complementary RNA or DNA strands. The new innovation in PNA coming from Heemstra’s PhD student