PMID- 30194891
OWN - NLM
STAT- MEDLINE
DCOM- 20190930
LR  - 20201217
IS  - 1439-7633 (Electronic)
IS  - 1439-4227 (Print)
IS  - 1439-4227 (Linking)
VI  - 19
IP  - 22
DP  - 2018 Nov 16
TI  - An RNase P-Based Assay for Accurate Determination of the 
      5'-Deoxy-5'-azidoguanosine-Modified Fraction of in Vitro-Transcribed RNAs.
PG  - 2353-2359
LID - 10.1002/cbic.201800447 [doi]
AB  - Chemoenzymatic approaches are important for generating site-specific, chemically 
      modified RNAs, a cornerstone for RNA structure-function correlation studies. T7 
      RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of a DNA template 
      containing the G-initiating class III Phi6.5 promoter is typically used to generate 
      5'-chemically modified RNAs by including a guanosine analogue (G analogue) 
      initiator in the IVT. However, the yield of 5'-G analogue-initiated RNA is often 
      poor and variable due to the high ratios of G analogue:GTP used in IVT. We 
      recently reported that a T7RNAP P266L mutant afforded an approximately three-fold 
      increase in fluorescent 5'-thienoguanosine-initiated pre-tRNA(Cys) compared to 
      the wild type T7RNAP. We have further explored the use of T7RNAP P266L to 
      generate 5'-deoxy-5'-azidoguanosine ((az) G)-initiated RNA and found that the 
      mutant yielded approximately four times more (az) G-initiated pre-tRNA(Cys) than 
      the wild type in an IVT containing a 10:1 ratio of (az) G:GTP. For accurate 
      quantitation of the 5'-(az) G-initiated RNA fraction, we employed RNase P, an 
      endonuclease that catalyzes the removal of the 5'-leader in pre-tRNAs. 
      Importantly, we show herein how RNase P can be leveraged for assessing 
      5'-G analogue incorporation in any RNA by rendering the target RNA, upon its 
      binding to a customized external guide sequence RNA, into an unnatural substrate 
      of RNase P. Such an approach in conjunction with T7RNAP P266L-based IVT should 
      aid chemoenzymatic methods that are designed to generate 5'-chemically modified 
      RNAs.
CI  - (c) 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
FAU - Lyon, Seth E
AU  - Lyon SE
AD  - Department of Chemistry and Biochemistry and, Center for RNA Biology, The Ohio 
      State University, 774 Biological Sciences Building, 484 West 12th Avenue, 
      Columbus, OH, 43210, USA.
AD  - Present address: Biological and Biomedical Sciences Graduate Program, Yale 
      University, New Haven, CT, 06520, USA.
FAU - Chen, Tien-Hao
AU  - Chen TH
AD  - Department of Chemistry and Biochemistry and, Center for RNA Biology, The Ohio 
      State University, 774 Biological Sciences Building, 484 West 12th Avenue, 
      Columbus, OH, 43210, USA.
AD  - Present address: Department of Physiology and Biophysics, Case Western Reserve 
      University, Cleveland, OH, 44106, USA.
FAU - Wallace, Andrew J
AU  - Wallace AJ
AD  - Department of Chemistry and Biochemistry and, Center for RNA Biology, The Ohio 
      State University, 774 Biological Sciences Building, 484 West 12th Avenue, 
      Columbus, OH, 43210, USA.
AD  - Present address: Department of Chemistry, University of California, Santa Cruz, 
      CA, 95094, USA.
FAU - Adib, Katie
AU  - Adib K
AD  - Department of Chemistry and Biochemistry and, Center for RNA Biology, The Ohio 
      State University, 774 Biological Sciences Building, 484 West 12th Avenue, 
      Columbus, OH, 43210, USA.
AD  - Present address: Wright State University Boonshoft School of Medicine, Dayton, 
      OH, 45435, USA.
FAU - Gopalan, Venkat
AU  - Gopalan V
AUID- ORCID: 0000-0002-3016-2719
AD  - Department of Chemistry and Biochemistry and, Center for RNA Biology, The Ohio 
      State University, 774 Biological Sciences Building, 484 West 12th Avenue, 
      Columbus, OH, 43210, USA.
LA  - eng
GR  - R01 GM120582/GM/NIGMS NIH HHS/United States
GR  - S10 OD023582/OD/NIH HHS/United States
GR  - T32 GM007223/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20181024
PL  - Germany
TA  - Chembiochem
JT  - Chembiochem : a European journal of chemical biology
JID - 100937360
RN  - 0 (RNA Precursors)
RN  - 0 (Viral Proteins)
RN  - 12133JR80S (Guanosine)
RN  - EC 2.7.7.- (bacteriophage T7 RNA polymerase)
RN  - EC 2.7.7.6 (DNA-Directed RNA Polymerases)
RN  - EC 3.1.26.5 (Ribonuclease P)
SB  - IM
MH  - DNA-Directed RNA Polymerases/*genetics
MH  - Guanosine/*analogs & derivatives
MH  - Mutation
MH  - Promoter Regions, Genetic
MH  - RNA Precursors/*genetics
MH  - Ribonuclease P/*genetics
MH  - *Transcription, Genetic
MH  - Viral Proteins/*genetics
PMC - PMC6392026
MID - NIHMS1521653
OTO - NOTNLM
OT  - 5' modification
OT  - RNA
OT  - azidoguanosine
OT  - in vitro transcription
COIS- The authors declare no competing financial interests.
EDAT- 2018/09/09 06:00
MHDA- 2019/10/01 06:00
PMCR- 2019/11/16
CRDT- 2018/09/09 06:00
PHST- 2018/08/02 00:00 [received]
PHST- 2018/09/09 06:00 [pubmed]
PHST- 2019/10/01 06:00 [medline]
PHST- 2018/09/09 06:00 [entrez]
PHST- 2019/11/16 00:00 [pmc-release]
AID - 10.1002/cbic.201800447 [doi]
PST - ppublish
SO  - Chembiochem. 2018 Nov 16;19(22):2353-2359. doi: 10.1002/cbic.201800447. Epub 2018 
      Oct 24.