RNA labeling, conjugation and ligation

Paredes, Eduardo; Evans, Molly; Das, Sauvik

doi:10.1016/j.ymeth.2011.02.008

Cited by 114 publications

(115 citation statements)

References 132 publications

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“…2, Scheme 3) [66–68]. In addition, labeling can also be achieved by post-transcription chemical reaction of an RNA carrying terminal functional groups with a fluorescent reagent [69,70]. For example, single labeling can be achieved by reaction of a thiol end-labeled RNA with commercially available fluorescent maleimide (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation

Zhang

Guo

2014

Methods

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Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The Single Molecule Photobleaching (SMPB) technology for direct counting developed by our team (Shu et al, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described.

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Section: Methodsmentioning

confidence: 99%

“…For example, single labeling can be achieved by reaction of a thiol end-labeled RNA with commercially available fluorescent maleimide (Fig. 2, Scheme 4) [69], or through “Click” chemistry of alkyne modified RNA with azide modified fluorophore [70–72]. …”

Section: Methodsmentioning

confidence: 99%

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation

Zhang

Guo

2014

Methods

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“…6 On the other hand, both chemical and enzymatic syntheses of modified RNA are more problematic and less well established, 7-9 despite the extensive current efforts in the use of modified RNA probes for imaging, chemical biology and therapeutic applications. 6,9,[10][11][12][13][14][15][16] The enzymatic production of modified RNAs can be based either on RNA polymerase catalyzed synthesis using modified ribonucleoside triphosphates (NTPs) or on enzymatic posttranscriptional modifications of RNA (e.g. alkylation by methyltransferases, etc.).…”

Section: Introductionmentioning

confidence: 99%

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates

et al. 2018

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We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides.Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.

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“…Although different chemical and enzymatic methods are available for labeling RNA at the 5’ or 3’ end (48), not all of them are compatible with native purification as they involve harsh conditions that could denature the RNA. Here we describe some of the labeling methods that may be used to generate a triply labeled, co-transcriptionally folded RNA sample.…”

Section: Introductionmentioning

confidence: 99%

Native Purification and Labeling of RNA for Single Molecule Fluorescence Studies

Rinaldi

Suddala

Walter

2014

RNA-RNA Interactions

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The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5’ end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.

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RNA labeling, conjugation and ligation

Cited by 114 publications

References 132 publications

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates

Native Purification and Labeling of RNA for Single Molecule Fluorescence Studies

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