Genes

Research

18 pages, 21909 KiB

Open AccessCommunication

Potential Misidentification of Natural Isomers and Mass-Analogs of Modified Nucleosides by Liquid Chromatography–Triple Quadrupole Mass Spectrometry

by Xiuying Lin, Qianhui Zhang, Yichao Qin, Qisheng Zhong, Daizhu Lv, Xiaopeng Wu, Pengcheng Fu and Huan Lin

Genes 2022, 13(5), 878; https://doi.org/10.3390/genes13050878 - 13 May 2022

Cited by 5 | Viewed by 4135

Abstract

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many [...] Read more.

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (>160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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23 pages, 3134 KiB

Open AccessArticle

Epitranscriptomic Reprogramming Is Required to Prevent Stress and Damage from Acetaminophen

by Sara Evke, Qishan Lin, Juan Andres Melendez and Thomas John Begley

Genes 2022, 13(3), 421; https://doi.org/10.3390/genes13030421 - 25 Feb 2022

Cited by 8 | Viewed by 2952

Abstract

Epitranscriptomic marks, in the form of enzyme catalyzed RNA modifications, play important gene regulatory roles in response to environmental and physiological conditions. However, little is known with respect to how acute toxic doses of pharmaceuticals influence the epitranscriptome. Here we define how acetaminophen [...] Read more.

Epitranscriptomic marks, in the form of enzyme catalyzed RNA modifications, play important gene regulatory roles in response to environmental and physiological conditions. However, little is known with respect to how acute toxic doses of pharmaceuticals influence the epitranscriptome. Here we define how acetaminophen (APAP) induces epitranscriptomic reprogramming and how the writer Alkylation Repair Homolog 8 (Alkbh8) plays a key gene regulatory role in the response. Alkbh8 modifies tRNA selenocysteine (tRNA^Sec) to translationally regulate the production of glutathione peroxidases (Gpx’s) and other selenoproteins, with Gpx enzymes known to play protective roles during APAP toxicity. We demonstrate that APAP increases toxicity and markers of damage, and decreases selenoprotein levels in Alkbh8 deficient mouse livers, when compared to wildtype. APAP also promotes large scale reprogramming of many RNA marks comprising the liver tRNA epitranscriptome including: 5-methoxycarbonylmethyluridine (mcm⁵U), isopentenyladenosine (i⁶A), pseudouridine (Ψ), and 1-methyladenosine (m¹A) modifications linked to tRNA^Sec and many other tRNA’s. Alkbh8 deficiency also leads to wide-spread epitranscriptomic dysregulation in response to APAP, demonstrating that a single writer defect can promote downstream changes to a large spectrum of RNA modifications. Our study highlights the importance of RNA modifications and translational responses to APAP, identifies writers as key modulators of stress responses in vivo and supports the idea that the epitranscriptome may play important roles in responses to pharmaceuticals. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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14 pages, 1543 KiB

Open AccessArticle

A Split NanoLuc Reporter Quantitatively Measures Circular RNA IRES Translation

by Priyanka Sehta, Ann-Marie Wilhelm, Shu-Jun Lin, Michelle A. Urman, Haley A. MacNeil and Gabriele Fuchs

Genes 2022, 13(2), 357; https://doi.org/10.3390/genes13020357 - 16 Feb 2022

Cited by 2 | Viewed by 4810

Abstract

Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can [...] Read more.

Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5′ untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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19 pages, 1798 KiB

Open AccessArticle

Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers

by Prabhat K. Mallik, Kimi Nishikawa, Pramit Mallik and Hua Shi

Genes 2022, 13(1), 86; https://doi.org/10.3390/genes13010086 - 29 Dec 2021

Cited by 2 | Viewed by 1976

Abstract

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, [...] Read more.

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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11 pages, 2006 KiB

Open AccessCommunication

Regulation and Site-Specific Covalent Labeling of NSUN2 via Genetic Encoding Expansion

by Jizhong Zhao, Hongmei Hu, Sheng Wang, Li Wang and Rui Wang

Genes 2021, 12(10), 1488; https://doi.org/10.3390/genes12101488 - 24 Sep 2021

Cited by 3 | Viewed by 2826

Abstract

In living organisms, RNA regulates gene expression, cell migration, differentiation, and cell death. 5-Methylcytosine is a post-transcriptional RNA modification in a wide range of RNA species, including messenger RNAs. The addition of m⁵C to RNA cytosines is enabled by the NSUN [...] Read more.

In living organisms, RNA regulates gene expression, cell migration, differentiation, and cell death. 5-Methylcytosine is a post-transcriptional RNA modification in a wide range of RNA species, including messenger RNAs. The addition of m⁵C to RNA cytosines is enabled by the NSUN enzyme family, a critical RNA methyltransferase. In this study, natural lysines modified with special groups were synthesized. Through two rounds of positive screening and one round of negative screening, we evaluated and identified the MbPylRS-tRNA_CUA unnatural lysine substitution system, which specifically recognizes lysine with a defined group. Moreover, non-natural lysine substitution at C271 of NSUN2 active site and the subsequent fluorescent labeling was realized through the click reaction. Then, the function of the NSUN2 mutant and its upregulated CDK1 gene as well as its effect on cell proliferation were evaluated. Efficient labeling and regulation of NSUN2 was achieved, laying the basis for further studies on the function and regulatory mechanism of upregulated genes. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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Review

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23 pages, 2748 KiB

Open AccessEditor’s ChoiceReview

Fluorescent Platforms for RNA Chemical Biology Research

by Jinxi Du, Ricky Dartawan, William Rice, Forrest Gao, Joseph H. Zhou and Jia Sheng

Genes 2022, 13(8), 1348; https://doi.org/10.3390/genes13081348 - 27 Jul 2022

Cited by 7 | Viewed by 3837

Abstract

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in [...] Read more.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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25 pages, 1714 KiB

Open AccessReview

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

by Rebecca J. D’Esposito, Christopher A. Myers, Alan A. Chen and Sweta Vangaveti

Genes 2022, 13(3), 540; https://doi.org/10.3390/genes13030540 - 18 Mar 2022

Cited by 8 | Viewed by 4234

Abstract

RNA is critical to a broad spectrum of biological and viral processes. This functional diversity is a result of their dynamic nature; the variety of three-dimensional structures that they can fold into; and a host of post-transcriptional chemical modifications. While there are many [...] Read more.

RNA is critical to a broad spectrum of biological and viral processes. This functional diversity is a result of their dynamic nature; the variety of three-dimensional structures that they can fold into; and a host of post-transcriptional chemical modifications. While there are many experimental techniques to study the structural dynamics of biomolecules, molecular dynamics simulations (MDS) play a significant role in complementing experimental data and providing mechanistic insights. The accuracy of the results obtained from MDS is determined by the underlying physical models i.e., the force-fields, that steer the simulations. Though RNA force-fields have received a lot of attention in the last decade, they still lag compared to their protein counterparts. The chemical diversity imparted by the RNA modifications adds another layer of complexity to an already challenging problem. Insight into the effect of RNA modifications upon RNA folding and dynamics is lacking due to the insufficiency or absence of relevant experimental data. This review provides an overview of the state of MDS of modified RNA, focusing on the challenges in parameterization of RNA modifications as well as insights into relevant reference experiments necessary for their calibration. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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10 pages, 1938 KiB

Open AccessEditor’s ChoiceReview

Recent Development of Prodrugs of Gemcitabine

by Bhoomika Pandit and Maksim Royzen

Genes 2022, 13(3), 466; https://doi.org/10.3390/genes13030466 - 5 Mar 2022

Cited by 35 | Viewed by 5285

Abstract

Gemcitabine is a nucleoside analog that has been used widely as an anticancer drug for the treatment of a variety of conditions, including ovarian, bladder, non-small-cell lung, pancreatic, and breast cancer. However, enzymatic deamination, fast systemic clearance, and the emergence of chemoresistance have [...] Read more.

Gemcitabine is a nucleoside analog that has been used widely as an anticancer drug for the treatment of a variety of conditions, including ovarian, bladder, non-small-cell lung, pancreatic, and breast cancer. However, enzymatic deamination, fast systemic clearance, and the emergence of chemoresistance have limited its efficacy. Different prodrug strategies have been explored in recent years, seeking to obtain better pharmacokinetic properties, efficacy, and safety. Different drug delivery strategies have also been employed, seeking to transform gemcitabine into a targeted medicine. This review will provide an overview of the recent developments in gemcitabine prodrugs and their effectiveness in treating cancerous tumors. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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19 pages, 3389 KiB

Open AccessReview

Advances in Therapeutic _L-Nucleosides and _L-Nucleic Acids with Unusual Handedness

by Yuliya Dantsu, Ying Zhang and Wen Zhang

Genes 2022, 13(1), 46; https://doi.org/10.3390/genes13010046 - 24 Dec 2021

Cited by 7 | Viewed by 4397

Abstract

Nucleic-acid-based small molecule and oligonucleotide therapies are attractive topics due to their potential for effective target of disease-related modules and specific control of disease gene expression. As the non-naturally occurring biomolecules, modified DNA/RNA nucleoside and oligonucleotide analogues composed of _L-(deoxy)riboses, have been [...] Read more.

Nucleic-acid-based small molecule and oligonucleotide therapies are attractive topics due to their potential for effective target of disease-related modules and specific control of disease gene expression. As the non-naturally occurring biomolecules, modified DNA/RNA nucleoside and oligonucleotide analogues composed of _L-(deoxy)riboses, have been designed and applied as innovative therapeutics with superior plasma stability, weakened cytotoxicity, and inexistent immunogenicity. Although all the chiral centers in the backbone are mirror converted from the natural _D-nucleic acids, _L-nucleic acids are equipped with the same nucleobases (A, G, C and U or T), which are critical to maintain the programmability and form adaptable tertiary structures for target binding. The types of _L-nucleic acid drugs are increasingly varied, from chemically modified nucleoside analogues that interact with pathogenic polymerases to nanoparticles containing hundreds of repeating _L-nucleotides that circulate durably in vivo. This article mainly reviews three different aspects of _L-nucleic acid therapies, including pharmacological _L-nucleosides, Spiegelmers as specific target-binding aptamers, and _L-nanostructures as effective drug-delivery devices. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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Other

Jump to: Research, Review

5 pages, 515 KiB

Open AccessPerspective

CRISPR-Guided Proximity Labeling of RNA–Protein Interactions

by Mingxing Lu, Zuowei Wang, Yixiu Wang and Bingbing Ren

Genes 2022, 13(9), 1549; https://doi.org/10.3390/genes13091549 - 27 Aug 2022

Cited by 1 | Viewed by 3583

Abstract

Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The resulting biotinylated proteins can then be isolated and identified. A combination of programmable DNA targeting and proximity labeling that maps [...] Read more.

Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The resulting biotinylated proteins can then be isolated and identified. A combination of programmable DNA targeting and proximity labeling that maps proteomic landscape at DNA elements with dCas9-APEX2 has been established in living cells. However, defining interactome at RNA elements has lagged behind. In combination with RNA-targeting CRISPR-Cas13, proximity labeling can also be used to identify proteins that interact with specific RNA elements in living cells. From this viewpoint, we briefly summarize the latest advances in CRISPR-guided proximity labeling in studying RNA–protein interactions, and we propose applying the most recent engineered proximity-labeling enzymes to study RNA-centric interactions in the future. Full article

(This article belongs to the Special Issue RNA Chemical Biology)

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