Efficient Chemical Syntheses of Long and Modified RNA Oligonucleotides Joel Myerson Agilent Technologies
Jul 28, 2015
Efficient Chemical Syntheses of Long and Modified RNA Oligonucleotides
Joel Myerson
Agilent Technologies
July 9, 2014
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Why synthetic RNA?
• Oligos containing modified natural or unnatural nucleotides
Stability
Specificity
Activity
Cross-linking
Labeling
• Oligos with well-defined ends
• Quantifiable mixtures with well-defined amounts
• Large amounts of RNA on the mg to gram scale
Synthetic RNA allows us to create:
ERCC 2.0 Joel Myerson
• Oligos of consistent purity
July 9, 2014
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• Orthogonal chemistry
→ High quality, biologically active RNA
• High efficiency per cycle
→ Routine synthesis of 120-mers
• Cost-effective monomers
→ Incorporation of modifications
• Simple product isolation
→ Suitable for high throughput
2’-Hydroxy 2’-Deoxy
TC-RNA chemistry Making RNA synthesis as robust and easy as DNA synthesis
ERCC 2.0 Joel Myerson
July 9, 2014
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TC- RNA synthesis
J. Am. Chem. Soc. 2011, 133, 11540–11556
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July 9, 2014
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RNA Problem #1
Need an extra protecting group on phosphoramidite monomer
Protected 2’-hydroxyl
RNA Synthesis Dogma #1
Cannot use a carbonyl protecting group because of 2’-3’-migration
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July 9, 2014
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RNA Problem #2
Unprotected RNA fragments under basic conditions
Base
RNA Synthesis Dogma #2
Cannot have a free 2’-OH in the presence of base
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July 9, 2014
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TC = the Thionocarbamate protecting group
TC = the 2´-O-(1, 1-dioxo1λ6-thiomorpholine-4-carbothioate) protecting group
TC-RNA Chemistry
July 9, 2014
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kobserved
Fraction
Cleaved
0.16 min-1
0.69
0.15 min-1
0.73
TC synthesized In vitro transcribed
Synthesize 49-mer enzyme strand of Schistosoma Mansoni Hammerhead ribozyme.
TC-RNA and in vitro transcribed RNA show the same activity
for substrate strand cleavage
Activity of synthetic TC-RNA
J.Swan, Agilent Technologies ; A. Pardi, University of Colorado, Boulder
July 9, 2014
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HPLC-ESI Mass Spectrometry to assess chemical purity
HPLC chromatogram of purified 49-mer Hammerhead Ribozyme
Mass spectra of 49-mer showing multiple
charge states
6 negative
charges
19 negative
charges
Deconvoluted mass spectra showing observed
mass of 15755.80. Calculated mass is 15755.62
“Extract” the average mass
spectra from the gray
region
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iROTS: Interoperable Robust Orthogonal Translation Systems
DARPA collaboration with Michael Jewett, Northwestern University
and Farren Isaacs, Yale University.
Calc30032.0
Obs30032.2
tRNA-amber natural (CUA anti-codon)
A260 m
AU
Calc26472.1
Obs26472.2
mRNA-amber natural (UAG codon)
HPLC chromatogram of crude
93-mer product
HPLC of purified 82-mer HPLC of purified 93-mer
Mass spectrum Mass spectrum
ERCC 2.0 Joel Myerson
July 9, 2014
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UUUAAGAAGGAGAUAUACAAUGGACUACAAAGACGACGACGACAAAAAAGGCisoGGCCGGGACCAUCAC
CAUCACCAUCACUGA
Calculated molecular weight = 26487.2
Observed molecular weight = 26486.9
Calculated molecular weight = 30047.2
Observed molecular weight = 30046.5
GGUGAGGUGGCCGAGAGGCUGAAGGCGCUCCCCUGCisoCAAGGGAGUAUGCGGUCAAAAGCUGCAUCCG
GGGUUCGAAUCCCCGCCUCACCGCCA
isoG
isoC
RNA oligos containing the orthogonal base pair iso-G and iso-C
iso-G mRNA
82-mer
iso-C tRNA
93-mer Mass spectrum
Mass spectrum
ERCC 2.0 Joel Myerson
iROTS: Interoperable Robust Orthogonal Translation Systems
DARPA collaboration with Michael Jewett, Northwestern University
and Farren Isaacs, Yale University.
July 9, 2014
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tRNA containing allylamino-U,
labeled with cy5-NHS ester
HPLC purified
Mass spectrum
ERCC 2.0 Joel Myerson
Labeling RNA for folding and structure studies
July 9, 2014
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Guide RNA
113-mer
AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGUUUAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
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CRISPR-Cas9
Mass =
36162.69 calculated
36162.84 observed 113-mer
mass spectrum
sgRNA CLTA-1
CLTA1
TC-RNA
Thank you to
J. Am. Chem. Soc. 2011, 133, 11540–11556
July 9, 2014 14
and
Jeff Swan
Ben Lunstad
Rob Kaiser
Neil Cook
Steve Laderman
Laurakay Bruhn
Agilent Technologies
and
Arthur Pardi
University of Colorado, Boulder
Michael Jewett
Northwestern University
Farren Isaacs
Yale University
ERCC 2.0 Joel Myerson