20140711 7 j_myerson_ercc2.0_workshop

Efficient Chemical Syntheses of Long and Modified RNA Oligonucleotides

Joel Myerson

Agilent Technologies

July 9, 2014

2

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

3

• 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


July 9, 2014

4

TC- RNA synthesis

J. Am. Chem. Soc. 2011, 133, 11540–11556


July 9, 2014

5

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


July 9, 2014

6

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


July 9, 2014


7

TC = the Thionocarbamate protecting group

TC = the 2´-O-(1, 1-dioxo1λ6-thiomorpholine-4-carbothioate) protecting group

TC-RNA Chemistry

July 9, 2014


8

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

9

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


July 9, 2014

10

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


July 9, 2014

11

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


iROTS: Interoperable Robust Orthogonal Translation Systems

DARPA collaboration with Michael Jewett, Northwestern University

and Farren Isaacs, Yale University.

July 9, 2014

12

tRNA containing allylamino-U,

labeled with cy5-NHS ester

HPLC purified

Mass spectrum


Labeling RNA for folding and structure studies

July 9, 2014

13

Guide RNA

113-mer

AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGUUUAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU


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


20140711 7 j_myerson_ercc2.0_workshop

Science

Transcript of 20140711 7 j_myerson_ercc2.0_workshop