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Article ; Online: Reactivity-based RNA profiling for analyzing transcriptome interactions of small molecules in human cells.

Fang, Linglan / Kool, Eric T

STAR protocols

2023 Volume 4, Issue 4, Page(s) 102670

Abstract: Protein-targeted small-molecule drugs may unintentionally bind intracellular RNA, contributing to drug toxicity. Moreover, new drugs are actively sought for intentionally targeting RNA. Here, we present a protocol to globally profile RNA-drug ... ...

Abstract	Protein-targeted small-molecule drugs may unintentionally bind intracellular RNA, contributing to drug toxicity. Moreover, new drugs are actively sought for intentionally targeting RNA. Here, we present a protocol to globally profile RNA-drug interactions in human cells using acylating probes and next-generation sequencing. We describe steps for cell culture, target acylation, library preparation, and sequencing. Detailed bioinformatic analyses identify drug-binding RNA loci in ∼16,000 poly(A)+ human transcripts. This streamlined workflow identifies RNA-drug interactions at single-nucleotide resolution, revealing widespread transcriptome interactions of drugs. For complete details on the use and execution of this protocol, please refer to Fang et al.
MeSH term(s)	Humans ; Transcriptome/genetics ; Acylation ; Cell Culture Techniques ; Computational Biology ; RNA/genetics
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2023-10-31
Publishing country	United States
Document type	Journal Article
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2023.102670
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Article: RNA Infrastructure Profiling Illuminates Transcriptome Structure in Crowded Spaces.

Xiao, Lu / Fang, Linglan / Kool, Eric T

bioRxiv : the preprint server for biology

2023

Abstract: RNAs can fold into compact three-dimensional structures, and most RNAs undergo protein interactions in the cell. These compact and occluded environments can block the ability of structure-probing agents to provide useful data about the folding and ... ...

Abstract	RNAs can fold into compact three-dimensional structures, and most RNAs undergo protein interactions in the cell. These compact and occluded environments can block the ability of structure-probing agents to provide useful data about the folding and modification of the underlying RNA. The development of probes that can analyze structure in crowded settings, and differentiate the proximity of interactions, can shed new light on RNA biology. To this end, here we employ 2'-OH-reactive probes that are small enough to access folded RNA structure underlying many close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. We compare reverse transcriptase stops in RNA-Seq data from probes of small and standard size to assess RNA-protein proximity and evaluate solvent-exposed tunnels adjacent to RNA. The data are analyzed first with structurally characterized complexes (human 18S and 28S RNA), and then applied transcriptome-wide to polyadenylated transcripts in HEK293 cells. In our transcriptome profile, the smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. We further show that acetyl probes provide superior signals for identifying m
Language	English
Publishing date	2023-10-09
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.10.09.561413
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Article ; Online: 2'-OH as a universal handle for studying intracellular RNAs.

Xiao, Lu / Fang, Linglan / Kool, Eric T

Cell chemical biology

2023 Volume 31, Issue 1, Page(s) 110–124

Abstract: RNA plays pivotal roles in most cellular processes, serving as both the traditional carrier of genetic information and as a key regulator of cellular functions. The advent of chemical technologies has contributed critically to the analysis of cellular ... ...

Abstract	RNA plays pivotal roles in most cellular processes, serving as both the traditional carrier of genetic information and as a key regulator of cellular functions. The advent of chemical technologies has contributed critically to the analysis of cellular RNA structures, functions, and interactions. Many of these methods and molecules involve the utilization of chemically reactive handles in RNAs, either introduced externally or inherent within the polymer itself. Among these handles, the 2'-hydroxyl (2'-OH) group has emerged as an exceptionally well-suited and general chemical moiety for the modification and profiling of RNAs in intracellular studies. In this review, we provide an overview of the recent advancements in intracellular applications of acylation at the 2'-OH group of RNA. We outline progress made in probing RNA structure and interactomes, controlling RNA function, RNA imaging, and analyzing RNA-small molecule interactions, all achieved in living cells through this simple chemical handle on the biopolymer.
MeSH term(s)	RNA/metabolism ; Nucleic Acid Conformation ; Acylation
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2023-11-21
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural
ISSN	2451-9448
ISSN (online)	2451-9448
DOI	10.1016/j.chembiol.2023.10.022
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Article ; Online: Efficient post-synthesis incorporation and conjugation of reactive ketones in RNA

Shioi, Ryuta / Xiao, Lu / Fang, Linglan / Kool, Eric T

Chemical communications (Cambridge, England)

2023 Volume 60, Issue 2, Page(s) 232–235

Abstract: Despite the broad utility of ketones in bioconjugation, few methods exist to introduce them into RNA. Here we develop highly reactive 2'-OH acylating reagents containing strained-ring ketones, and employ them as versatile labeling handles for RNA. ...

Abstract	Despite the broad utility of ketones in bioconjugation, few methods exist to introduce them into RNA. Here we develop highly reactive 2'-OH acylating reagents containing strained-ring ketones, and employ them as versatile labeling handles for RNA.
MeSH term(s)	Ketones ; RNA/genetics ; Acylation ; Indicators and Reagents
Chemical Substances	Ketones ; RNA (63231-63-0) ; Indicators and Reagents
Language	English
Publishing date	2023-12-21
Publishing country	England
Document type	Journal Article
ZDB-ID	1472881-3
ISSN	1364-548X ; 1359-7345 ; 0009-241X
ISSN (online)	1364-548X
ISSN	1359-7345 ; 0009-241X
DOI	10.1039/d3cc05123d
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Article ; Online: Acylation probing of "generic" RNA libraries reveals critical influence of loop constraints on reactivity.

Xiao, Lu / Fang, Linglan / Kool, Eric T

Cell chemical biology

2022 Volume 29, Issue 8, Page(s) 1341–1352.e8

Abstract: The reactivity of RNA 2'-OH acylation is broadly useful both in probing structure and in preparing conjugates. To date, this reactivity has been analyzed in limited sets of biological RNA sequences, leaving open questions of how reactivity varies ... ...

Abstract	The reactivity of RNA 2'-OH acylation is broadly useful both in probing structure and in preparing conjugates. To date, this reactivity has been analyzed in limited sets of biological RNA sequences, leaving open questions of how reactivity varies inherently without regard to sequence in structured contexts. We constructed and probed "generic" structured RNA libraries using homogeneous loop sequences, employing deep sequencing to carry out a systematic survey of reactivity. We find a wide range of RNA reactivities among single-stranded sequences, with nearest neighbors playing substantial roles. Remarkably, certain small loops are found to be far more reactive on average (up to 4,000-fold) than single-stranded RNAs, due to conformational constraints that enhance reactivity. Among loops, we observe large variations in reactivity based on size, type, and position. The results lend insights into RNA designs for achieving high-efficiency local conjugation and provide new opportunities to refine structure analysis.
MeSH term(s)	Acylation ; Gene Library ; Nucleic Acid Conformation ; RNA/metabolism
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2022-06-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	2451-9448
ISSN (online)	2451-9448
DOI	10.1016/j.chembiol.2022.05.005
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Article ; Online: The Right Tool for the Job: A Chemical and Genetic Toolkit for Interrogating DCLK1 Function.

Fang, Linglan / Maly, Dustin J

Cell chemical biology

2020 Volume 27, Issue 10, Page(s) 1221–1223

Abstract: Cell permeable, small molecule inhibitors are powerful tools for interrogating kinase function and validating drug targets. In this issue of Cell Chemical Biology, Liu and colleagues (2020) describe the development of a toolkit containing a highly ... ...

Abstract	Cell permeable, small molecule inhibitors are powerful tools for interrogating kinase function and validating drug targets. In this issue of Cell Chemical Biology, Liu and colleagues (2020) describe the development of a toolkit containing a highly selective DCLK1 inhibitor and complementary DCLK1 mutants for interrogating DCLK1-dependent cellular processes.
MeSH term(s)	Biology ; Protein-Serine-Threonine Kinases/genetics ; Protein-Serine-Threonine Kinases/metabolism ; RNA Processing, Post-Transcriptional
Chemical Substances	Protein-Serine-Threonine Kinases (EC 2.7.11.1)
Language	English
Publishing date	2020-10-29
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Comment
ISSN	2451-9448
ISSN (online)	2451-9448
DOI	10.1016/j.chembiol.2020.09.007
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Article ; Online: Diverse Reagent Scaffolds Provide Differential Selectivity of 2'-OH Acylation in RNA.

Xiao, Lu / Fang, Linglan / Chatterjee, Sayantan / Kool, Eric T

Journal of the American Chemical Society

2022 Volume 145, Issue 1, Page(s) 143–151

Abstract: RNA 2'-OH acylation is widely used both for mapping structure and for conjugating RNA, generally relying on selective reactions with unpaired nucleotides over paired ones. Common reagents for this acylation have been chiefly restricted to two similar ... ...

Abstract	RNA 2'-OH acylation is widely used both for mapping structure and for conjugating RNA, generally relying on selective reactions with unpaired nucleotides over paired ones. Common reagents for this acylation have been chiefly restricted to two similar aryl scaffolds, leaving open the question of how more broadly varied reagent structure might affect selectivity. Here, we prepared a set of 10 structurally diverse acylimidazole reagents and employed deep sequencing to profile their reactivity and selectivity in an RNA library of systematically varied structure. We show that structure-directed reactivity profiles vary significantly with the reagent scaffold, and we document new acylating agents that have altered selectivity profiles, including reagents that show elevated selectivity within loops, as well as compounds with reduced off-target reactivity in loop closing base pairs. Interestingly, we also show that the simplest reagent (acetylimidazole) is cell permeable and is small enough to map RNA structure in the presence of protein contacts that block other reagents. Finally, we describe reagents that show elevated selectivity within small loops, with applications in site-selective labeling. The results provide new tools for improved conjugation and mapping of RNA.
MeSH term(s)	RNA/chemistry ; Indicators and Reagents ; Nucleic Acid Conformation ; Base Pairing ; Acylation
Chemical Substances	RNA (63231-63-0) ; Indicators and Reagents
Language	English
Publishing date	2022-12-21
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.2c09040
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Article ; Online: Reversible 2'-OH acylation enhances RNA stability.

Fang, Linglan / Xiao, Lu / Jun, Yong Woong / Onishi, Yoshiyuki / Kool, Eric T

Nature chemistry

2023 Volume 15, Issue 9, Page(s) 1296–1305

Abstract: The presence of a hydroxyl group at the 2'-position in its ribose makes RNA susceptible to hydrolysis. Stabilization of RNAs for storage, transport and biological application thus remains a serious challenge, particularly for larger RNAs that are not ... ...

Abstract	The presence of a hydroxyl group at the 2'-position in its ribose makes RNA susceptible to hydrolysis. Stabilization of RNAs for storage, transport and biological application thus remains a serious challenge, particularly for larger RNAs that are not accessible by chemical synthesis. Here we present reversible 2'-OH acylation as a general strategy to preserve RNA of any length or origin. High-yield polyacylation of 2'-hydroxyls ('cloaking') by readily accessible acylimidazole reagents effectively shields RNAs from both thermal and enzymatic degradation. Subsequent treatment with water-soluble nucleophilic reagents removes acylation adducts quantitatively ('uncloaking') and recovers a remarkably broad range of RNA functions, including reverse transcription, translation and gene editing. Furthermore, we show that certain α-dimethylamino- and α-alkoxy- acyl adducts are spontaneously removed in human cells, restoring messenger RNA translation with extended functional half-lives. These findings support the potential of reversible 2'-acylation as a simple and general molecular solution for enhancing RNA stability and provide mechanistic insights for stabilizing RNA regardless of length or origin.
MeSH term(s)	Humans ; RNA/chemistry ; Proteins/metabolism ; Water ; Acylation ; RNA, Messenger/genetics ; Indicators and Reagents
Chemical Substances	RNA (63231-63-0) ; Proteins ; Water (059QF0KO0R) ; RNA, Messenger ; Indicators and Reagents
Language	English
Publishing date	2023-06-26
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2464596-5
ISSN	1755-4349 ; 1755-4330
ISSN (online)	1755-4349
ISSN	1755-4330
DOI	10.1038/s41557-023-01246-6
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Article ; Online: Pervasive transcriptome interactions of protein-targeted drugs.

Fang, Linglan / Velema, Willem A / Lee, Yujeong / Xiao, Lu / Mohsen, Michael G / Kietrys, Anna M / Kool, Eric T

Nature chemistry

2023 Volume 15, Issue 10, Page(s) 1374–1383

Abstract: The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as ... ...

Abstract	The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off-targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling methodology and applied it to uncover transcriptome interactions of a set of Food and Drug Administration-approved small-molecule drugs in vivo. We show that these protein-targeted drugs pervasively interact with the human transcriptome and can exert unintended biological effects on RNA functions. In addition, we show that many off-target interactions occur at RNA loci associated with protein binding and structural changes, allowing us to generate hypotheses to infer the biological consequences of RNA off-target binding. The results suggest that rigorous characterization of drugs' transcriptome interactions may help assess target specificity and potentially avoid toxicity and clinical failures.
Language	English
Publishing date	2023-08-31
Publishing country	England
Document type	Journal Article
ZDB-ID	2464596-5
ISSN	1755-4349 ; 1755-4330
ISSN (online)	1755-4349
ISSN	1755-4330
DOI	10.1038/s41557-023-01309-8
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Article ; Online: Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Chakraborty, Sujata / Ahler, Ethan / Simon, Jessica J / Fang, Linglan / Potter, Zachary E / Sitko, Katherine A / Stephany, Jason J / Guttman, Miklos / Fowler, Douglas M / Maly, Dustin J

Cell chemical biology

2023 Volume 31, Issue 2, Page(s) 207–220.e11

Abstract: Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively ... ...

Abstract	Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.
MeSH term(s)	src-Family Kinases/genetics ; Catalytic Domain ; Phosphorylation ; Adenosine Triphosphate/metabolism ; Drug Resistance
Chemical Substances	src-Family Kinases (EC 2.7.10.2) ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2023-09-07
Publishing country	United States
Document type	Journal Article
ISSN	2451-9448
ISSN (online)	2451-9448
DOI	10.1016/j.chembiol.2023.08.005
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