From the journal RSC Chemical Biology Peer review history

09-May-2022

Dear Dr Brown:

Manuscript ID: CB-ART-04-2022-000100
TITLE: Covalently attached intercalators restore duplex stability and splice-switching activity to triazole-modified oligonucleotides

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I look forward to receiving your revised manuscript.

Yours sincerely,
Andrea (Rentmeister)

************

Reviewer 1

The research included in the manuscript is aimed at adressing key challenges of the antisense field, ie. biostability and biodistribution. Triazole-modified oligonucleotides are synthesized with interchalators / guanidino groups attached to the 2'-O position of one of the nucleosides of the triazole-linked dimer units.
Tm binding studies are conducted showing that the affinity-decreasing effect of the triazole linkage in part can be counterbalanced by a pyrene (Py) or anthraquinone (Aq) interchalator attached with the best effects observed when binding to DNA targets. This is somewhat unfortunate as RNA is the relevant biological target.
In particular against RNA the Aq binding is better than the Py binding - probably intercalation is most relevant for the Py analogs and this is known to be difficult into RNA. This aspect and why the pyrene was selected for biological studies should be discussed carefully in a revised version of the manuscript.
In order not to overstate the biological results it should be highlighted that the obtained splice switching data are at best similar and in most cases not as good as for the controls ON19 and ON20.
An alternative design would be to use more simple, eg. DNA-DNA triazole-linked dimers, and then add intercalators (using known modifications) at one or more of the monomeric nucleotides. It is unclear to this referee why the present design was selected and arguments in relation to this are suggested to be added.
In short the manuscript is potentially interesting but with respect to design background and limited effects on RNA binding, in vivo biology data would be needed to increase impact and general interest. Therefore submission to another journal, eg. OBC, is recommended.

Reviewer 2

In this article, the authors report an interesting method for the improvement of duplex stability and restoration of antisense activity of oligonucleotides equipped with triazole linkages instead of canonical phosphodiesters. To do so, the authors installed a 2’-amino linker on a ribose and on the 5’-side of a triazole linkage. The resulting phosphoramidite dimers were compatible with solid phase DNA and RNA synthesis. The amine acted as a synthetic anchor to append additional functional groups aiming at improving the stability of duplexes including pyrene, anthraquinone, and guanidine. Functionalized oligonucleotides were subjected to a thorough UV melting analysis which revealed that pyrene and anthraquinone modifications restored (and even increased) duplex stability when compared to unmodified DNA, while guanidine had no detectable effect. This stabilizing effect was also observed in 2’-OMe/PS containing oligonucleotides which were then used in two different types of splice switching assays. The pyrene modified sequences restored the antisense effect in these assays. All experiments have been carried out in a competent manner, the manuscript is well written, and the supporting information is of very good overall quality. Taken together, this manuscript demonstrates a novel method for increasing duplex stability and restoring antisense activity which could be used for other types of sugar, backbone, and/or nucleobase-modified oligonucleotides. Publication in RSC Chemical Biology is therefore recommended pending some (minor) revisions:
- The authors should provide a tentative explanation on the fact that the guanidine modification did not result in any stabilizing effect.
- It would be interesting to see whether such a modification pattern could be introduced into gapmers and whether the resulting constructs could recruit RNase H. While I do not insist on such additional experiments, these would further increase the quality of the present study.
- The authors could cite more recent reviews on the introduction of chemical modifications into ASOs (see e.g. JBC 2021, 296, 100416; Chem Soc Rev 2021, 50, 5126).

We thank the referees for their careful and insightful comments. We have taken all of them into account in the revised manuscript, and our detailed point by point response is below.

We look forward to hearing from you.

Sincerely,

Tom Brown, Professor of Nucleic Acid Chemistry.


REVIEWER REPORT(S):

Referee: 1

Comments to the Author
The research included in the manuscript is aimed at adressing key challenges of the antisense field, ie. biostability and biodistribution. Triazole-modified oligonucleotides are synthesized with interchalators / guanidino groups attached to the 2'-O position of one of the nucleosides of the triazole-linked dimer units.
Tm binding studies are conducted showing that the affinity-decreasing effect of the triazole linkage in part can be counterbalanced by a pyrene (Py) or anthraquinone (Aq) interchalator attached with the best effects observed when binding to DNA targets. This is somewhat unfortunate as RNA is the relevant biological target.

The referee is correct in stating that stabilisation of triazole-pyrene or anthraquinone against DNA is a little greater than it is against RNA. However, the oligonucleotide backbone in these comparative melting studies (in table 1) is deoxyribose-phosphodiester (natural DNA). This means that the duplex is an RNA-DNA hybrid. In the cell studies in the paper, one cannot use an unmodified DNA backbone as it is essential to use a modified backbone that is stable in cells. For this reason, we used a 2’-O-methyl RNA-phosphorothioate backbone which is known to favour RNA targets over DNA targets, so selectivity for RNA over DNA should not be an issue. We have explained this in the revised manuscript (page 4 column 1). An additional point, as there are far more copies of RNA than DNA within a cell we are not too concerned by the affinity for DNA, especially given that DNA is often tied up (histones/enzymes/double stranded) and the increase in stability is not enough to trigger strand invasion.

In particular against RNA the Aq binding is better than the Py binding - probably intercalation is most relevant for the Py analogs and this is known to be difficult into RNA. This aspect and why the pyrene was selected for biological studies should be discussed carefully in a revised version of the manuscript.

The relevant data is in Table 2 of the paper, and the discussion is at the bottom of page 4 column 2, i.e.:
“The stabilising effect of AQ and PY with triple and quadruple additions of the triazole linkage was not as great as anticipated. Adding PY to ON11 with three triazole linkages only increased the Tm by 5.0 oC (ON14), and the resulting duplex was 9.7 oC less stable than the control (ON19). ON15 with four PY functionalised triazole linkages was only 7.3 oC more stable than ON12 without PY, and 13.5 oC less stable than the control. More surprisingly, addition of three and four anthraquinone moieties next to triazole linkages (ON17 Tm = 51.1 oC and ON18 Tm = 45.1 oC) caused a decrease in duplex stability compared to the triazole containing ON11 (Tm = 55.5 oC) and ON12 (Tm = 49.4 oC).

From the melting studies on 2’-OMe-modified oligonucleotides against RNA, which are more relevant to the cell studies than the ones in Table 1, we decided to pursue pyrene and not anthraquinone.

To emphasise this, we have changed the text on page 5 left column from:

“Having confirmed that PY is capable of partially compensating for the loss of duplex stability incurred with multiple triazoles in PS/2′-OMe ASOs, we evaluated efficacy of the modified ASOs in a commonly used splice-switching reporter cell system.46”
To:
“Having confirmed that PY is more capable than anthraquinone of partially compensating for the loss of duplex stability incurred with multiple triazoles in PS/2′-OMe ASOs, we evaluated efficacy of the modified ASOs in a commonly used splice-switching reporter cell system.46”

In order not to overstate the biological results it should be highlighted that the obtained splice switching data are at best similar and in most cases not as good as for the controls ON19 and ON20.

This is a good point. We have mentioned this in the revised manuscript (conclusions, changes underlined):
“As anticipated, the triazole alone resulted in lower activity; however, addition of PY to the ONs fully or partially restored the lost activity and, in some cases, the combination resulted in more potent ASO than the parent ASO with a standard 2’-OMe-phosphorothioate backbone.”

An alternative design would be to use more simple, eg. DNA-DNA triazole-linked dimers, and then add intercalators (using known modifications) at one or more of the monomeric nucleotides. It is unclear to this referee why the present design was selected and arguments in relation to this are suggested to be added.
The reason that the pyrene and anthraquinone were placed at the triazole site is the following:
It is known that these aromatic groups stabilise unmodified duplexes due to intercalation or groove binding. However, in this study, we specifically wanted to find out if they could confer duplex stability when placed directly next to an artificial backbone. This is not known, so our study contributes new information to the literature. We have added this discussion to the revised manuscript (page 2 column 2).

In short the manuscript is potentially interesting but with respect to design background and limited effects on RNA binding, in vivo biology data would be needed to increase impact and general interest. Therefore submission to another journal, eg. OBC, is recommended.

We believe that we have suitably addressed the referee’s comments, and the revised manuscript is improved because of this. We submitted the paper in response to an invitation from RSC Chemical Biology for the themed collection: ‘XNA: Xeno-nucleic acids’ and we would prefer it to be published as part of this themed collection.


Referee: 2

Comments to the Author
In this article, the authors report an interesting method for the improvement of duplex stability and restoration of antisense activity of oligonucleotides equipped with triazole linkages instead of canonical phosphodiesters. To do so, the authors installed a 2’-amino linker on a ribose and on the 5’-side of a triazole linkage. The resulting phosphoramidite dimers were compatible with solid phase DNA and RNA synthesis. The amine acted as a synthetic anchor to append additional functional groups aiming at improving the stability of duplexes including pyrene, anthraquinone, and guanidine. Functionalized oligonucleotides were subjected to a thorough UV melting analysis which revealed that pyrene and anthraquinone modifications restored (and even increased) duplex stability when compared to unmodified DNA, while guanidine had no detectable effect. This stabilizing effect was also observed in 2’-OMe/PS containing oligonucleotides which were then used in two different types of splice switching assays. The pyrene modified sequences restored the antisense effect in these assays. All experiments have been carried out in a competent manner, the manuscript is well written, and the supporting information is of very good overall quality. Taken together, this manuscript demonstrates a novel method for increasing duplex stability and restoring antisense activity which could be used for other types of sugar, backbone, and/or nucleobase-modified oligonucleotides. Publication in RSC Chemical Biology is therefore recommended pending some (minor) revisions:

We thank the referee for their supportive and positive comments.

- The authors should provide a tentative explanation on the fact that the guanidine modification did not result in any stabilizing effect.

This is a good point. Guanidines are cationic at neutral pH and are known to stabilise unmodified duplexes by masking charge repulsion between opposite anionic nucleic acid strands. Because the cationic guanidine is close to an uncharged triazole linkage in the modified duplexes, stabilisation does not occur as there is no ‘anion-anion’ charge repulsion at this locus in the duplex. To be honest, when designing the study, we did expect some stabilisation to occur due to the guanidine due to its influence on the broader unmodified region of the duplex, but it did not. We have added this discussion to the manuscript (Page 3 right column).

- It would be interesting to see whether such a modification pattern could be introduced into gapmers and whether the resulting constructs could recruit RNase H. While I do not insist on such additional experiments, these would further increase the quality of the present study.

This is a good suggestion, but as the referee suggests, we feel that it is outside the scope of the current study. It is certainly worth exploring in future. Placing the modification in the gap has a low probability of success due to the stringent structural/helical requirements of RNase-H. However, placing the modification in the wings of antisense oligonucleotides is also worth exploring as this should not interfere with the activity of RNase-H.
We have added this discussion to the manuscript, penultimate paragraph of the conclusions.

- The authors could cite more recent reviews on the introduction of chemical modifications into ASOs (see e.g. JBC 2021, 296, 100416; Chem Soc Rev 2021, 50, 5126).

We thank the referee for these suggestions. These additional references have been added to the revised manuscript.

15-May-2022

Dear Dr Brown:

Manuscript ID: CB-ART-04-2022-000100.R1
TITLE: Covalently attached intercalators restore duplex stability and splice-switching activity to triazole-modified oligonucleotides

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Andrea (Rentmeister)

Reviewer 1

It's the opinion of this referee that the changes made in the revised version have improved the manuscript by securing a better balance in the statements and a more relevant positioning within the context of the field.