15. Coupling of boronic esters 6 and 7 with RNA transcript 13
using catalyst Pd(OAc) 2 (L1) 2 gave two regioisomers (major
trans and minor cis) of the RNA product [29]. When Pd
(OAc) 2 (L2) 2 was used a single isomer (trans) was formed.
However, the overall yield of the coupled RNA product was
better when the reaction was performed using Pd(OAc) 2 (L1) 2 .
Therefore, depending on the boronic acid/ester substrate, we
recommend the users of this protocol to optimize the conditions in terms of reaction time and type of catalyst that can
be used.
16. Mass of oligonucleotides can be measured using Applied Biosystems 4800 Plus MALDI TOF/TOF analyzer.
Acknowledgments
This work was supported by Wellcome Trust-DBT India Alliance
(IA/S/16/1/502360) grant to S.G.S. M.B.W. thanks CSIR,
India, for a graduate research fellowship. The authors wish to
thank Arun Tanpure for discussion and help with work that has
led to the optimization of this protocol.
References
1. Prescher JA, Bertozzi CR (2005) Chemistry in
living systems. Nat Chem Biol 1:13–21
2. Weisbrod SH, Marx A (2011) Novel strategies
for the site-specific covalent labelling of nucleic
acids. Chem Commun 47:7018
3. Spicer CD, Davis BG (2014) Selective chemical
protein modification. Nat Commun 5:4740
4. Holstein JM, Rentmeister A (2016) Current
covalent modification methods for detecting
RNA in fixed and living cells. Methods
98:18–25
5. McKay CS, Finn MG (2014) Click chemistry in
complex mixtures: bioorthogonal bioconjugation. Chem Biol 21:1075–1101
6. van Berkel SS, van Eldijk MB, van Hest JCM
(2011) Staudinger ligation as a method for
bioconjugation. Angew Chem Int Ed
50:8806–8827
7. Wu H, Devaraj NK (2016) Inverse electrondemand Diels-Alder bioorthogonal reactions.
Top Curr Chem 374:3
8. George JT, Srivatsan SG (2017) Posttranscriptional chemical labeling of RNA by using
bioorthogonal chemistry. Methods 120:28–38
9. El-Sagheer AH, Brown T (2010) New strategy
for the synthesis of chemically modified RNA
constructs exemplified by hairpin and
hammerhead ribozymes. Proc Natl Acad Sci
U S A 107:15329–15334
10. Rao H, Tanpure AA, Sawant AA, Srivatsan SG
(2012) Enzymatic incorporation of an azidemodified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization. Nat Protoc 7:1097–1112
11. Samanta A, Krause A, J€ aschke A (2014) A
modified dinucleotide for site-specific RNA-labelling by transcription priming and click
chemistry. Chem Commun 50:1313–1316
12. Someya T, Ando A, Kimoto M, Hirao I (2015)
Site-specific labeling of RNA by combining
genetic alphabet expansion transcription and
copper-free click chemistry. Nucleic Acids Res
43:6665–6676
13. Holstein JM, Stummer D, Rentmeister A
(2016) Enzymatic modification of 5’-capped
RNA and subsequent labeling by click chemistry. Methods Mol Biol 428:45–60
14. Jao CY, Salic A (2008) Exploring RNA transcription and turnover in vivo by using click
chemistry. Proc Natl Acad Sci U S A
105:15779–15784
15. Grammel M, Hang H, Conrad NK (2012)
Chemical reporters for monitoring RNA synthesis and poly (A) tail dynamics. Chembiochem 13:1112–1115
Labeling RNA for Conformational Analysis and Imaging
485
using catalyst Pd(OAc) 2 (L1) 2 gave two regioisomers (major
trans and minor cis) of the RNA product [29]. When Pd
(OAc) 2 (L2) 2 was used a single isomer (trans) was formed.
However, the overall yield of the coupled RNA product was
better when the reaction was performed using Pd(OAc) 2 (L1) 2 .
Therefore, depending on the boronic acid/ester substrate, we
recommend the users of this protocol to optimize the conditions in terms of reaction time and type of catalyst that can
be used.
16. Mass of oligonucleotides can be measured using Applied Biosystems 4800 Plus MALDI TOF/TOF analyzer.
Acknowledgments
This work was supported by Wellcome Trust-DBT India Alliance
(IA/S/16/1/502360) grant to S.G.S. M.B.W. thanks CSIR,
India, for a graduate research fellowship. The authors wish to
thank Arun Tanpure for discussion and help with work that has
led to the optimization of this protocol.
References
1. Prescher JA, Bertozzi CR (2005) Chemistry in
living systems. Nat Chem Biol 1:13–21
2. Weisbrod SH, Marx A (2011) Novel strategies
for the site-specific covalent labelling of nucleic
acids. Chem Commun 47:7018
3. Spicer CD, Davis BG (2014) Selective chemical
protein modification. Nat Commun 5:4740
4. Holstein JM, Rentmeister A (2016) Current
covalent modification methods for detecting
RNA in fixed and living cells. Methods
98:18–25
5. McKay CS, Finn MG (2014) Click chemistry in
complex mixtures: bioorthogonal bioconjugation. Chem Biol 21:1075–1101
6. van Berkel SS, van Eldijk MB, van Hest JCM
(2011) Staudinger ligation as a method for
bioconjugation. Angew Chem Int Ed
50:8806–8827
7. Wu H, Devaraj NK (2016) Inverse electrondemand Diels-Alder bioorthogonal reactions.
Top Curr Chem 374:3
8. George JT, Srivatsan SG (2017) Posttranscriptional chemical labeling of RNA by using
bioorthogonal chemistry. Methods 120:28–38
9. El-Sagheer AH, Brown T (2010) New strategy
for the synthesis of chemically modified RNA
constructs exemplified by hairpin and
hammerhead ribozymes. Proc Natl Acad Sci
U S A 107:15329–15334
10. Rao H, Tanpure AA, Sawant AA, Srivatsan SG
(2012) Enzymatic incorporation of an azidemodified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization. Nat Protoc 7:1097–1112
11. Samanta A, Krause A, J€ aschke A (2014) A
modified dinucleotide for site-specific RNA-labelling by transcription priming and click
chemistry. Chem Commun 50:1313–1316
12. Someya T, Ando A, Kimoto M, Hirao I (2015)
Site-specific labeling of RNA by combining
genetic alphabet expansion transcription and
copper-free click chemistry. Nucleic Acids Res
43:6665–6676
13. Holstein JM, Stummer D, Rentmeister A
(2016) Enzymatic modification of 5’-capped
RNA and subsequent labeling by click chemistry. Methods Mol Biol 428:45–60
14. Jao CY, Salic A (2008) Exploring RNA transcription and turnover in vivo by using click
chemistry. Proc Natl Acad Sci U S A
105:15779–15784
15. Grammel M, Hang H, Conrad NK (2012)
Chemical reporters for monitoring RNA synthesis and poly (A) tail dynamics. Chembiochem 13:1112–1115
Labeling RNA for Conformational Analysis and Imaging
485