5′-End Labeling of RNA with [γ-<sup>32</sup>P]ATP and T4 Polynucleotide Kinase

Rio, Donald C.

doi:10.1101/pdb.prot080739

Cited by 18 publications

(6 citation statements)

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“…For already-existing RNA strands in particular, including those from living systems, few practical chemical methods are available for functionalization. Although labeling at remote 5′ and 3′ ends is feasible, − functionalization of the internal nucleotides of RNA has received little attention until very recently (Scheme ). Given the complexities and biomedical importance of RNA biology, the elucidation of new reactivities for this biopolymer could provide useful tools for labeling and analysis in a biological setting.…”

mentioning

confidence: 99%

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

et al. 2019

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Acylation of RNA at 2′-OH groups is widely applied in mapping RNA structure and recently for controlling RNA function. Reactions are described that install the smallest 2-carbon acyl groups on RNA—namely, 2′-O-acetyl and 2′-O-carbonate groups. Hybridization and thermal melting experiments are performed to assess the effects of the acyl groups on duplex formation. Both reagents can be employed at lower concentrations to map RNA secondary structure by reverse transcriptase primer extension (SHAPE) methods.

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confidence: 99%

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

et al. 2019

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“…In the end, the biophysical characterization of quantitative and qualitative LPIs may be achieved via the application of methods such as the electrophoretic mobility shift assay (EMSA) [ 62 ], filter-binding assays [ 62 ], or surface plasmon resonance [ 63 , 64 ].…”

Section: Current Methods For the Functional Analysis Of Lncrnasmentioning

confidence: 99%

“…Cross-linking immunoprecipitation (CLIP) [49,50] Targets of RNA-binding proteins identified by editing (TRIBE) [51] Digestion-optimized RNA immunoprecipitation cDNA library sequencing (DO-RIP-seq) [52] RNA-affinity purification followed by mass spectrometry [53][54][55][56] RNA-affinity purification followed by protein microarrays [57] The isolation of target RNA molecules by biotinylated antisense probes [54,58] The isolation of target RNA molecules by peptide nucleic acid oligomers [55] HB-tag-based affinity RNA purification [56] Chromatin isolation with RNA purification (ChIRP) [59,60] RNA chromosome conformation capture (R3C) [61] LPI computational prediction [72] The biophysical characterization of quantitative and qualitative LPIs Electrophoretic mobility shift assay (EMSA) [62] Filter-binding assays [82] Surface plasmon resonance [63,64] The evaluation of the coding capacity of lncRNAs…”

Section: Determination Of the Intracellular Lncrna Localizationmentioning

confidence: 99%

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management

Melone

Salvati

Brusco

et al. 2023

IJMS

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In the complex and articulated machinery of the human genome, less than 2% of the transcriptome encodes for proteins, while at least 75% is actively transcribed into non-coding RNAs (ncRNAs). Among the non-coding transcripts, those ≥200 nucleotides long (lncRNAs) are receiving growing attention for their involvement in human diseases, particularly cancer. Genomic studies have revealed the multiplicity of processes, including neoplastic transformation and tumor progression, in which lncRNAs are involved by regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels by mechanism(s) that still need to be clarified. In breast cancer, several lncRNAs were identified and demonstrated to have either oncogenic or tumor-suppressive roles. The functional understanding of the mechanisms of lncRNA action in this disease could represent a potential for translational applications, as these molecules may serve as novel biomarkers of clinical use and potential therapeutic targets. This review highlights the relationship between lncRNAs and the principal hallmark of the luminal breast cancer phenotype, estrogen receptor α (ERα), providing an overview of new potential ways to inhibit estrogenic signaling via this nuclear receptor toward escaping resistance to endocrine therapy.

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“…97 One can quantify the bound versus unbound RNA by comparing the RNA-protein band versus the apo RNA band, respectively. Specificity can be evaluated through the addition of unlabeled control RNA, which would not be expected to impact complex formation, as well as through the addition of unlabeled RNA target, which should displace the labeled RNA in a dose-dependent manner.…”

Section: Biophysical Characterization Of Lncrna:protein Complexesmentioning

confidence: 99%

“…Electrophoretic mobility shift assay (EMSA), also known as a gel shift assay or band shift assay, is an affinity-based technique that can be used to quantitatively or qualitatively study RNA-protein interactions based on the size difference between apo and protein-bound states of RNA . One can quantify the bound versus unbound RNA by comparing the RNA-protein band versus the apo RNA band, respectively.…”

Section: Biophysical Characterization Of Lncrna:protein Complexesmentioning

confidence: 99%

Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin

McFadden

Hargrove

2016

Biochemistry

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Long noncoding RNAs (lncRNAs), defined as non-translated transcripts greater than 200 nucleotides in length, are often differentially expressed throughout developmental stages, tissue types, and disease states. The identification, visualization, and suppression/overexpression of these sequences have revealed impacts on a wide range of biological processes, including epigenetic regulation. Biochemical investigations on select systems have revealed striking insight into the biological roles of lncRNAs and lncRNA:protein complexes, which in turn prompt even more unanswered questions. To begin, multiple protein-and RNA-centric technologies have been employed to isolate lncRNA:protein and lncRNA:chromatin complexes. LncRNA interactions with the multi-subunit protein complex PRC2, which acts as a transcriptional silencer, represent some of the few cases where the binding affinity, selectivity, and activity of a lncRNA:protein complex have been investigated. At the same time, recent reports of full-length lncRNA secondary structures suggest the formation of complex structures with multiple independent folding domains and pave the way for more detailed structural investigations and predictions of lncRNA threedimensional structure. This review will provide an overview of the methods and progress made to date as well as highlight new methods that promise to further inform the molecular recognition, specificity, and function of lncRNAs. Graphical Abstract *Corresponding Author: amanda.hargrove@duke.edu. Notes The authors declare no competing financial interest. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe "noncoding RNA revolution" (Cech, Stetiz) has revealed myriad functional RNA molecules with roles extending far beyond that of a messenger between DNA and protein. 1 The world of noncoding RNAs (ncRNAs), or RNAs that are not usually translated to proteins, came to light in large part as a result of the Encyclopedia of DNA Elements (ENCODE) project. 2 This consortium found that while up to 90% of the genome was transcribed only 1.2% was translated to protein. Furthermore, this large pool of untranslated transcripts demonstrated biochemical indices of function traditionally ascribed solely to proteins. 3 Research exploring the biological activity of these ncRNA transcripts promptly grew. Among the many newly discovered functions of noncoding RNAs, several classes are now known to play critical roles in the regulation of gene expression 1 as well as disease progression. 4 NcRNAs are classified based on size, with small ncRNAs less than 200 n.t. and long noncoding RNAs (lncRNAs) greater than 200 n.t. Several small ncRNA classes, including microRNAs (miRNAs) and small-interfering RNAs (siRNA), regulate gene expression by forming partially complementary duplexes with mRNAs, which in turn promote mRNA degradation or inhibit mRNA translation into peptides. [5][6][7][8] LncRNAs, on the other hand, have been found to exhibit a wide range of regulatory roles, including traffick...

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5′-End Labeling of RNA with [γ-³²P]ATP and T4 Polynucleotide Kinase

Cited by 18 publications

References 5 publications

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management

Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin

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5′-End Labeling of RNA with [γ-32P]ATP and T4 Polynucleotide Kinase

Cited by 18 publications

References 5 publications

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management

Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin

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5′-End Labeling of RNA with [γ-³²P]ATP and T4 Polynucleotide Kinase