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Article
Effects of Chemically Modified Messenger RNA on Protein Expression Bin Li, Xiao Luo, and Yizhou Dong Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00090 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 12
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Bioconjugate Chemistry
Effects of Chemically Modified Messenger RNA on Protein Expression
1 2 3
Bin Li,†,§ Xiao Luo,†,§ and Yizhou Dong*,†,‡,¶,
4
†
5
Columbus, Ohio 43210, USA
6
‡
7
¶
8
USA
9
Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University,
Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA
The Center for Clinical and Translational Science, The Ohio State University, Columbus, Ohio 43210,
The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA
10 11 12 13
ABSTRACT
14
Chemically modified nucleotides play significant roles in the effectiveness of messenger RNA
15
(mRNA) translation. Here, we describe the synthesis of two sets of chemically modified mRNAs
16
[encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively],
17
evaluation of protein expression, and correlation analysis of expression level under various conditions.
18
The results indicate chemical modifications of mRNAs are able to significantly improve protein
19
expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with
20
N1-methylpseudouridine (me1ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications
21
ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than
22
other eGFP mRNAs. Consequently, me1ψ, 5moU, and ψ are promising nucleotides for chemical
23
modification of mRNAs.
24 25 26 27 28 29 30 1
ACS Paragon Plus Environment
Bioconjugate Chemistry
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31
Page 2 of 12
Messenger RNAs (mRNAs) encoding functional proteins have demonstrated their therapeutic in
fundamental
and
clinical
studies.1-5
32
potential
For
example,
immunotherapy
with
33
mRNA-electroporated dendritic cell provided therapeutic benefit in several cancer clinical trials.4,6
34
mRNAs were also utilized to produce chimeric antigen receptors in T cells for adoptive T-cell
35
therapy,7 to express functional proteins for protein replacement therapy,8-12 and most recently, to make
36
nucleases for gene engineering.13,14 Although there have been significant advances in mRNA-based
37
therapeutics in the past decade, instability and immunogenicity of mRNA hinders its therapeutic
38
application in humans.1,4,5,15,16
39
In order to address these issues, numerous strategies for mRNA modification have been
40
investigated to improve translation efficiency and reduce immunogenicity, including modifications at
41
the 5’ cap, 5’ and 3’-untranslated regions, the coding region, and the poly(A) tail.1 Incorporation of
42
chemically modified nucleotides into mRNAs (Figure 1a) is one of the effective approaches reported
43
in the literature.1 For instance, Karikó et al. showed that pseudouridine (ψ) modified mRNA increased
44
expression of encoded erythropoietin.17,18 Kormann et al. reported that combination of 2-thiouridine
45
(s2U) and 5-methylcytidine (5meC) in modified mRNAs extended expression of encoded protein to
46
four weeks.11 Recently, Zangi et al. achieved induction of vascular regeneration using modified
47
(5meC and ψ) mRNA encoding human vascular endothelial growth factor-A.10 Consistently, Uchida et
48
al. stated that combination of 5meC and ψ in mRNAs augmented protein expression.19 Most recently,
49
Andries et al. reported that N1-methylpseudouridine (me1ψ) modified mRNA enhanced luciferase
50
expression and reduced immunogenicity.20 These studies demonstrate the importance of chemically
51
modified nucleotides on mRNA structural optimization. However, our knowledge of structure-activity
52
relationship on chemically modified mRNAs remains limited. Herein, we report synthesis and
53
evaluation of a library of chemically modified mRNAs. We conducted a correlation analysis for
54
protein expression of mRNAs at various conditions including translation temperature, cell types, and
55
coding sequences. Importantly, we identified me1ψ, 5-methoxyuridine (5moU) and ψ as favorable
56
nucleotides for improving protein expression.
57
2
ACS Paragon Plus Environment
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
a)
Expression Unmodified mRNA
Expression Chemically Modified mRNA
Protein
b) NH2
O
NH2
NH2
N3
H
O
N
N
NH2 HO
N
NH2
N
N
HO
2
6
N1 R
O
N O R 5-formylcytidine (5fC)
Cytidine (C) O 4
O
N R
O
5-methylcytidine (5meC)
O
NH
H
5-methoxycytidine (5moC)
O NH
O
N R
N1 R
O
N O R 5-formyluridine (5fU)
Uridine (U) NH2
7
N
5
NH
NH
9N R
4
N O R 5-methyluridine (5meU)
N1
N
N
N 3
Adenosine (A)
N R
6
N O R 5-carboxymethylesteruridine (5camU)
N
N6-methyladenosine (me6A)
N 3
R
N
NH
O R pseudouridine ( )
O R N1-methylpseudouridine (me1 )
O NH
S 4
9
O NH
1
NH
8
8 2
5
O HN
NH
O 7
N O R 5-hydroxymethylcytidine (5hmC)
O
O
N O R 5-methoxyuridine (5moU)
HN
6
5-hydroxycytidine (5hoC)
O
2
6
O
N R
O O
3
5
58
NH2
4
5
2
NH2
Guanosine (G)
N
NH2
R = ribose
R Thienoguanosine (thG)
59 60
Figure 1. (a) Illustration: chemically modified mRNAs improve protein expression. (b) A panel of
61
chemically modified nucleotides utilized for in vitro transcription, from which 24 mRNAs were
62
produced, including single modifications and combination of modifications. Blue: natural nucleotides.
63 64
In order to investigate the effects of chemical modifications on mRNAs, we first synthesized a
65
library of 24 firefly Luciferase mRNAs (FLuc mRNAs) using chemically modified nucleotides
66
(Figure 1b). We incorporated substitution of single modifications and combinations of modifications.
67
We then evaluated FLuc expression of mRNAs in a rabbit reticulocyte lysate system at 30 °C
68
according to the manufacturer's protocol. As shown in Figure 2a, eight of twenty four FLuc mRNAs
69
showed higher luciferase intensity compared to unmodified FLuc mRNA (three single modifications:
70
ψ, 5moU, me1ψ; and five combination modifications: 5hmC/5meU, 5meC/ψ, 5moC/5meU,
71
5meC/5moU, and 5meC/me1ψ). Among them, ψ-modified FLuc mRNA displayed the highest increase
72
of translation efficiency. To study the effects of temperature on mRNA translation, we further 3
ACS Paragon Plus Environment
Bioconjugate Chemistry
73
conducted the experiment at 37 °C. Analysis of the results indicated significant correlation for
74
translation at 30 and 37 °C (p
Article
Effects of Chemically Modified Messenger RNA on Protein Expression Bin Li, Xiao Luo, and Yizhou Dong Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00090 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
Effects of Chemically Modified Messenger RNA on Protein Expression
1 2 3
Bin Li,†,§ Xiao Luo,†,§ and Yizhou Dong*,†,‡,¶,
4
†
5
Columbus, Ohio 43210, USA
6
‡
7
¶
8
USA
9
Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University,
Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA
The Center for Clinical and Translational Science, The Ohio State University, Columbus, Ohio 43210,
The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA
10 11 12 13
ABSTRACT
14
Chemically modified nucleotides play significant roles in the effectiveness of messenger RNA
15
(mRNA) translation. Here, we describe the synthesis of two sets of chemically modified mRNAs
16
[encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively],
17
evaluation of protein expression, and correlation analysis of expression level under various conditions.
18
The results indicate chemical modifications of mRNAs are able to significantly improve protein
19
expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with
20
N1-methylpseudouridine (me1ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications
21
ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than
22
other eGFP mRNAs. Consequently, me1ψ, 5moU, and ψ are promising nucleotides for chemical
23
modification of mRNAs.
24 25 26 27 28 29 30 1
ACS Paragon Plus Environment
Bioconjugate Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
31
Page 2 of 12
Messenger RNAs (mRNAs) encoding functional proteins have demonstrated their therapeutic in
fundamental
and
clinical
studies.1-5
32
potential
For
example,
immunotherapy
with
33
mRNA-electroporated dendritic cell provided therapeutic benefit in several cancer clinical trials.4,6
34
mRNAs were also utilized to produce chimeric antigen receptors in T cells for adoptive T-cell
35
therapy,7 to express functional proteins for protein replacement therapy,8-12 and most recently, to make
36
nucleases for gene engineering.13,14 Although there have been significant advances in mRNA-based
37
therapeutics in the past decade, instability and immunogenicity of mRNA hinders its therapeutic
38
application in humans.1,4,5,15,16
39
In order to address these issues, numerous strategies for mRNA modification have been
40
investigated to improve translation efficiency and reduce immunogenicity, including modifications at
41
the 5’ cap, 5’ and 3’-untranslated regions, the coding region, and the poly(A) tail.1 Incorporation of
42
chemically modified nucleotides into mRNAs (Figure 1a) is one of the effective approaches reported
43
in the literature.1 For instance, Karikó et al. showed that pseudouridine (ψ) modified mRNA increased
44
expression of encoded erythropoietin.17,18 Kormann et al. reported that combination of 2-thiouridine
45
(s2U) and 5-methylcytidine (5meC) in modified mRNAs extended expression of encoded protein to
46
four weeks.11 Recently, Zangi et al. achieved induction of vascular regeneration using modified
47
(5meC and ψ) mRNA encoding human vascular endothelial growth factor-A.10 Consistently, Uchida et
48
al. stated that combination of 5meC and ψ in mRNAs augmented protein expression.19 Most recently,
49
Andries et al. reported that N1-methylpseudouridine (me1ψ) modified mRNA enhanced luciferase
50
expression and reduced immunogenicity.20 These studies demonstrate the importance of chemically
51
modified nucleotides on mRNA structural optimization. However, our knowledge of structure-activity
52
relationship on chemically modified mRNAs remains limited. Herein, we report synthesis and
53
evaluation of a library of chemically modified mRNAs. We conducted a correlation analysis for
54
protein expression of mRNAs at various conditions including translation temperature, cell types, and
55
coding sequences. Importantly, we identified me1ψ, 5-methoxyuridine (5moU) and ψ as favorable
56
nucleotides for improving protein expression.
57
2
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Page 3 of 12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
a)
Expression Unmodified mRNA
Expression Chemically Modified mRNA
Protein
b) NH2
O
NH2
NH2
N3
H
O
N
N
NH2 HO
N
NH2
N
N
HO
2
6
N1 R
O
N O R 5-formylcytidine (5fC)
Cytidine (C) O 4
O
N R
O
5-methylcytidine (5meC)
O
NH
H
5-methoxycytidine (5moC)
O NH
O
N R
N1 R
O
N O R 5-formyluridine (5fU)
Uridine (U) NH2
7
N
5
NH
NH
9N R
4
N O R 5-methyluridine (5meU)
N1
N
N
N 3
Adenosine (A)
N R
6
N O R 5-carboxymethylesteruridine (5camU)
N
N6-methyladenosine (me6A)
N 3
R
N
NH
O R pseudouridine ( )
O R N1-methylpseudouridine (me1 )
O NH
S 4
9
O NH
1
NH
8
8 2
5
O HN
NH
O 7
N O R 5-hydroxymethylcytidine (5hmC)
O
O
N O R 5-methoxyuridine (5moU)
HN
6
5-hydroxycytidine (5hoC)
O
2
6
O
N R
O O
3
5
58
NH2
4
5
2
NH2
Guanosine (G)
N
NH2
R = ribose
R Thienoguanosine (thG)
59 60
Figure 1. (a) Illustration: chemically modified mRNAs improve protein expression. (b) A panel of
61
chemically modified nucleotides utilized for in vitro transcription, from which 24 mRNAs were
62
produced, including single modifications and combination of modifications. Blue: natural nucleotides.
63 64
In order to investigate the effects of chemical modifications on mRNAs, we first synthesized a
65
library of 24 firefly Luciferase mRNAs (FLuc mRNAs) using chemically modified nucleotides
66
(Figure 1b). We incorporated substitution of single modifications and combinations of modifications.
67
We then evaluated FLuc expression of mRNAs in a rabbit reticulocyte lysate system at 30 °C
68
according to the manufacturer's protocol. As shown in Figure 2a, eight of twenty four FLuc mRNAs
69
showed higher luciferase intensity compared to unmodified FLuc mRNA (three single modifications:
70
ψ, 5moU, me1ψ; and five combination modifications: 5hmC/5meU, 5meC/ψ, 5moC/5meU,
71
5meC/5moU, and 5meC/me1ψ). Among them, ψ-modified FLuc mRNA displayed the highest increase
72
of translation efficiency. To study the effects of temperature on mRNA translation, we further 3
ACS Paragon Plus Environment
Bioconjugate Chemistry
73
conducted the experiment at 37 °C. Analysis of the results indicated significant correlation for
74
translation at 30 and 37 °C (p
