Fluorogenic Aptasensors with Small Molecules
Round 1
Reviewer 1 Report
This manuscript overviews the recent advances in duplexed or molecular beacon-Type fluorogenic aptasensors, fluorogenic aptazymes for biosensing or bioimaging. The current limitations and promising opportunities to promote the development of fluorogenic aptasensors are discussed briefly.
- Recent advances in fluorogenic aptasensor have been reviewed by the authors and summarized in this manuscript, most of them are only simple description and a critical evaluation of these previous works is not sufficient.
- The introduction section for small molecules is insufficient. The authors should make a further elaboration on the small molecules and conventional analytical methods for small molecules in the introduction section.
- The last section needs more comprehensive discussions for current limitations, and where has research to, in order to solve these limitations?
- The authors should check the reference styles (ref. 7, 20, 21, 25).
Author Response
Response to Reviewer #1
This manuscript overviews the recent advances in duplexed or molecular beacon-Type fluorogenic aptasensors, fluorogenic aptazymes for biosensing or bioimaging. The current limitations and promising opportunities to promote the development of fluorogenic aptasensors are discussed briefly.
1. Recent advances in fluorogenic aptasensor have been reviewed by the authors and summarized in this manuscript, most of them are only simple description and a critical evaluation of these previous works is not sufficient.
Our response: Thank you for the helpful comment. This review contains 99 references, so only the important points were summarized and described. As the reviewer pointed out, to criticize the previous works, we described common limitations and current challenges in the conclusion section in our revised manuscript.
2. The introduction section for small molecules is insufficient. The authors should make a further elaboration on the small molecules and conventional analytical methods for small molecules in the introduction section.
Our response: Thank you for the helpful comment. As the reviewer suggested, we added further description on small molecules in the introduction section in our revised manuscript (lines 33-38).
3. The last section needs more comprehensive discussions for current limitations, and where has research to, in order to solve these limitations?
Our response: Thank you for the helpful comment. As the review pointed out, we added more comprehensive discussions to the conclusion section in our revised manuscript.
4. The authors should check the reference styles (ref. 7, 20, 21, 25).
Our response: Thank you for pinpointing the wrong part. We revised the format of references according to the journal’s guideline (Ref. 7, 20, 21, 25, 43, 50, 51, 53, 61, 63, 69, 70, 88, 92, 95, 98, and 102).
Reviewer 2 Report
This review article summarizes some examples of applying fluorophore-modified aptamers for the detection and cellular imaging of small molecules and endogenous RNAs. It appears useful for the community associated with the design of aptamer-based nanostructure as a fluorescent probe. However, this review article still has room to be improved. First of all, long ncRNA is a macromolecule; thus, the title of "Fluorogenic Aptasensors with Small Molecules" should be modified. Secondly, this review article will be impressive if the authors describe the quenching mechanism between the quencher and fluorophore-modified aptamer and FRET mechanism fluorophore-modified aptamer and the other fluorophore-modified DNA. Thirdly, the authors did not discuss these reported nanoprobes' applications to detect target analytes in real-world samples. Fourthly, the authors did not clarify the difference between this review article and the previous review articles associated with aptamer-based sensors. Fifthly, the authors did not include the detection of small molecules using a coralyne-based aptamer probe. Lastly, the final part of this review article covers the fluorescent imaging of RNAs and metabolites. Some interesting observations are presented. Please show some image examples of cellular imaging of target analytes using an aptamer-based probe. I recommend the publication of this well-written and well-organized manuscript after a minor revision has been made.
Author Response
Response to Reviewer #2
This review article summarizes some examples of applying fluorophore-modified aptamers for the detection and cellular imaging of small molecules and endogenous RNAs. It appears useful for the community associated with the design of aptamer-based nanostructure as a fluorescent probe. However, this review article still has room to be improved. I recommend the publication of this well-written and well-organized manuscript after a minor revision has been made.
1. First of all, long ncRNA is a macromolecule; thus, the title of "Fluorogenic Aptasensors with Small Molecules" should be modified.
Our response: Thank you for the helpful comment. In this review, we highlighted three different aptasensors with focusing on small molecules as their targets. However, as the reviewer pointed out, we mentioned non-coding RNAs (miRNA or LncRNA) as the macromolecular target in the case of the fluorogen-activating aptamer reporters (Section 4 and Table 1). This is because most references use the RNA aptamer reporters to detect non-coding RNAs as well as small molecules, where measurement principle and riboswitch-type function are shared. Despite the macromolecular target, we think it is not appropriate to change the title to cover only a few cases in the overall context. Instead, we added this explanation on target molecules to our revised manuscript (lines 314-317).
2. Secondly, this review article will be impressive if the authors describe the quenching mechanism between the quencher and fluorophore-modified aptamer and FRET mechanism fluorophore-modified aptamer and the other fluorophore-modified DNA.
Our response: Thank you for the helpful comment. As the reviewer suggested, we added the further description on FRET and quenching mechanisms to our revised manuscript (lines 73-78).
3. Thirdly, the authors did not discuss these reported nanoprobes' applications to detect target analytes in real-world samples.
Our response: Thank you for the helpful comment. As the reviewer suggested, we added the further discussion on real samples to the conclusion section in our revised manuscript (lines 439-449).
4. Fourthly, the authors did not clarify the difference between this review article and the previous review articles associated with aptamer-based sensors.
Our response: Thank you for the helpful comment. As the reviewer mentioned, it is important to avoid the redundancy with other review articles. To do this, as clearly stated in the introduction, we highlight innovative fluorogenic aptamers based on small molecule-induced structural changes, while the previous review articles on aptamers have relied on fluorescence (FL)- and/or affinity-based assays or images without smart control of FL.
5. Fifthly, the authors did not include the detection of small molecules using a coralyne-based aptamer probe.
Our response: Thank you for the helpful comment. As the reviewer suggested, we described aptasensors using coralyne-triggered dsDNA in our revised manuscript with additional references (lines 110-115).
6. Lastly, the final part of this review article covers the fluorescent imaging of RNAs and metabolites. Some interesting observations are presented. Please show some image examples of cellular imaging of target analytes using an aptamer-based probe.
Our response: Thank you for the helpful comment. As the reviewer suggested, we modified Figure 3 by inserting metabolite imaging data into Figure 3D in our revised manuscript.
Reviewer 3 Report
Dear author(s),
This is an excellent review of the subject. It has been written clearly and concise. Also it is very useful for anyone working in this field.
One remark: at page 1, line 28: you mention high stability compared to antibodies. As far as I know, antibodies may be very stable. With regard ssDNA/RNA-molecules, should there be taken strict precautions to reach this high stability (buffers, temperature, etc.)?
Page 2, line 51: could you explain the phrase 'genetically encoded RNA'.
Page 2 line 62: maybe you better use 'discuss' instead of 'discussed'.
Page 3: at this page many abbrevations are used. Could please write terms at full when using the first time.
Page 6, line 237: I assume that you mean 'units' instead of 'unis'.
Page 6, line 260: maybe you better use 'a higher signal...' instead of 'a more signal...'.
Page 6, line 273-274: shouldn't 'an fluorogenic detection' be 'a fluorogenic detection method'?
Further, this manuscript is ready for publication.
Author Response
Response to Reviewer #3
This is an excellent review of the subject. It has been written clearly and concise. Also it is very useful for anyone working in this field.
One remark:
at page 1, line 28: you mention high stability compared to antibodies. As far as I know, antibodies may be very stable. With regard ssDNA/RNA-molecules, should there be taken strict precautions to reach this high stability (buffers, temperature, etc.)?
Our response: Thank you for the helpful comment. As the reviewer suggested, we modified “stability” to “thermal stability” to clarify the meaning.
Page 2, line 51: could you explain the phrase 'genetically encoded RNA'.
Our response: Thank you for the helpful comment. Similar to genetically encoded proteins, genetically encoded RNAs consist of two parts; in vitro selected RNA sequences and their cognate small molecule fluorogens. These fluorogens are cell permeable and nonfluorescent in the free state and their binding with the corresponding RNA sequences enhances their fluorescence by several orders of magnitude. This is called as genetically encoded RNA aptamers or light-up RNA aptamers. We described this explanation in our revised manuscript (lines 302-305).
Page 2 line 62: maybe you better use 'discuss' instead of 'discussed'.
Our response: We corrected the word, as the reviewer suggested.
Page 3: at this page many abbrevations are used. Could please write terms at full when using the first time.
Our response: Thank you for the helpful comment. As the reviewer suggested, we added the full names of all abbreviations when the word first appears.
Page 6, line 237: I assume that you mean 'units' instead of 'unis'.
Page 6, line 260: maybe you better use 'a higher signal...' instead of 'a more signal...'.
Page 6, line 273-274: shouldn't 'an fluorogenic detection' be 'a fluorogenic detection method'?
Our response: Thank you for pinpointing the grammar errors. In the revised manuscript, we revised those words as well as entire sentences with the assistance of professional English correction company. The corrected parts are marked in RED.
Reviewer 4 Report
The authors present a comprehensive review of recent advances in fluorogenic aptasensors in response to small molecules and provide broad applications of fluorogenic aptamers in biosensors and bioimaging. However, it is necessary to improve the discussion of current limitations and future work, giving possible strategies to overcome some of the remaining challenges in aptamer design, sensor development and small molecule diagnostics, especially in real samples.
Author Response
Response to Reviewer #4
The authors present a comprehensive review of recent advances in fluorogenic aptasensors in response to small molecules and provide broad applications of fluorogenic aptamers in biosensors and bioimaging. However, it is necessary to improve the discussion of current limitations and future work, giving possible strategies to overcome some of the remaining challenges in aptamer design, sensor development and small molecule diagnostics, especially in real samples.
Our response: Thank you for the helpful comment. As the reviewer suggested, we added the further discussions on limitations, challenges, overcoming strategies, and future aspects to the conclusion section in our revised manuscript (lines 439-453).
