Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry

George, Jerrin Thomas; Srivatsan, Seergazhi G.

doi:10.1016/j.ymeth.2017.02.004

Cited by 33 publications

(32 citation statements)

References 131 publications

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“…However, limited permeability and selectivity of the antibodies and compromised function of aptamer-tagged RNA have been the downsides of these methods. Alternatively, bio-orthogonal chemical reactions provide an easy route to label RNA in vitro and cellular milieu for a variety of applications 11–15. In this approach, a reactive group is introduced into an RNA ON by either chemical, enzymatic (RNA polymerases), or chemo-enzymatic (transferases) means, and further bioconjugation to label RNA is achieved by performing a chemoselective reaction with a cognate reactive partner containing a desired biophysical reporter.…”

Section: Introductionmentioning

confidence: 99%

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA

George

Srivatsan

2017

Bioconjugate Chem.

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The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions. Furthermore, we show for the first time the use of a palladium-mediated oxidative Heck reaction in functionalizing RNA with fluorogenic probes by reacting vinyl-labeled RNA transcripts with appropriate boronic acid substrates. The vinyl label also permitted the post-transcriptional functionalization of RNA by a reagent-free inverse electron demand Diels-Alder (IEDDA) reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the incorporation of VUTP provides newer possibilities for the modular functionalization of RNA with variety of reporters.

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Section: Introductionmentioning

confidence: 99%

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA

George

Srivatsan

2017

Bioconjugate Chem.

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“…These strategies usually use modified nucleoside phosphoramidites or triphosphates, which involve elaborate chemical manipulations. Moreover, in several instances, the substrates (i) show poor coupling efficiency, (ii) do not survive the conditions used in the solid-phase protocols and (iii) are not efficiently incorporated by polymerases ( 4 ). In this context, post-transcriptional coupling of IU-labeled RNA ONs with heterocycle-containing boronic acids/esters should provide direct access to RNA functionalized with responsive nucleoside probes.…”

Section: Resultsmentioning

confidence: 99%

“…phosphoramidites and triphosphates) and challenges associated with their incorporation (e.g. stability under reaction conditions, poor coupling and enzymatic incorporation efficiency) limit the applications of these methods ( 4 ). In this context, post-synthetic modification of RNA by using bioorthogonal reactions is proving as a valuable tool to generate functional RNA probes.…”

Section: Introductionmentioning

confidence: 99%

“…In this method, an RNA ON is labeled with a small reactive handle by using solid-phase ON synthesis protocol or by using the substrate promiscuity of RNA polymerases and certain RNA processing enzymes (e.g. transferases, 4–6 ). Following this step, a chemoselective reaction with the cognate reactive partner is performed to introduce the desired functional modification into the RNA.…”

Section: Introductionmentioning

confidence: 99%

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Post-transcriptional labeling by using Suzuki–Miyaura cross-coupling generates functional RNA probes

Walunj

Tanpure

Srivatsan

2018

Nucleic Acids Research

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Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki–Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki–Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

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“…6 Posttranscriptional chemical modifications through bioorthogonal chemistry is another alternative approach to modified RNA. 17,18 The enzymatic synthesis of modified RNA relies almost exclusively on the use of bacteriophage T7 RNA polymerase to avoid the need for complex transcription factors. The T7 RNA polymerase requires specific promoters [19][20][21] and the presence of guanosines in the +1 and/or +2 positions to ensure efficient transcription initiation.…”

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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Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry

Cited by 33 publications

References 131 publications

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA

Post-transcriptional labeling by using Suzuki–Miyaura cross-coupling generates functional RNA probes

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

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