From Selection to Use: Aptamers as Targeting Reagents in Hematology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provides a comprehensive and cell-centric review of aptamer applications in hematology, effectively highlighting both the potential and current limitations in the field. The organization by specific blood cell lineages (B cells, T cells, NK cells, and red blood cells) is a valuable approach that clearly exposes the biases and gaps in current aptamer development, such as the concentration on diseased cells over healthy counterparts and a narrow range of target markers. The discussion of SELEX methodologies and aptamer applications in cell isolation, therapeutics, and biosensing is thorough and well-supported by the provided references. The following are two minor comments and suggestions for improvement:

• Table 1 (Comparison of properties): The description for the "Availability long-term" attribute appears to be correctly placed under each category (Aptamers vs. Monoclonal antibodies) within the manuscript text. 
• Table 2 (Reported aptamers): For greater transparency and utility for future research and reproducibility, the nucleotide sequences of all listed aptamers should be added to the table. 

Overall, the manuscript presents a clear, insightful, and valuable contribution to the literature on aptamers in hematology, effectively defining the current landscape and future opportunities.

Author Response

See file

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review presents developed aptamers by cell lineages rather than diseases or technological platforms, specifically examining aptamers for B cells, T cells, natural killer cells, and red blood cells. The author evaluated reported applications, design strategies, and experimental use cases alongside persistent limitations in target selection and biological resolution. The review is organized by cell lineage rather than disease, highlighting biological challenges, offering a unique perspective, and clearly pointing out current research biases (such as an excessive focus on malignant cells and a lack of research on healthy cells), which is enlightening. Before considering its publication, the following issues need to be addressed.

1. Although Table 1 lists comparative items in terms of size, stability, modification, and so on, it does not delve into the pharmacokinetic differences of aptamers in the blood environment.

2. Table 2 only lists some targets. It is recommended to supplement the list or indicate that it is an example.

3. The article mentions that the coagulation target aptamer has entered clinical trials, but it does not discuss its successful experiences and lessons learned from failures.

4. Some sentences are too long (such as the abstract and introduction sections), and it is recommended to split them to enhance readability.

5. The format of references needs to be unified (some citations are not fully annotated).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The presented manuscript is a critical review of aptamer literature in hematology. The authors used a very interesting approach and arranged their review according to blood cell lineages. Such an unconventional method made it possible to draw attention to existing limitations in the application and selection of aptamers for blood cell recognition.

The writing and organization of the manuscript are excellent. It includes enough illustrations and pertinent citations.

The authors highlighted the benefits and limitations of aptamers and suggested several methods for better aptamer validation and characterization. I have several comments for authors that would improve the manuscript.

1. The text appears to be confused in the line "availability long-term" of Table 1 (page 5).
2. Figure 2 was not mentioned in the text, as far as I could see. The authors might want to double-check this.
3. I think Table 2's references need to be revised. It seems a little excessive to use both kinds of references at the same time.
4. If the authors included the K_D measuring method (SPR, BLI, fluorescence etc.) in Table 2, I think it would be highly helpful for future readers.
5. I suggest moving Figure 3 to Section 4.1. In this instance, the figure will be located beside the appropriate piece of text.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript deals with an interesting topic with a distinct orientation from other previous reports but, in my opinion, the potential of this topic is not fully exploited.

1. For example. Table 2 is central in this ms but it is quite simplistic. Authors acknowledge the lack of standardized aptamer literature that compromises the reliability of the aptamer performance under real conditions and contributes to delay in implementation.
   1. Table 2 must compile the complete sequences of aptamers used instead of just stating the nature (RNA or DNA) and the modification (please revise, some are not disclosed, e.g.  ref 82) and if they were truncated, multimerized or combined with other aptamer for bivalency. This could help the researcher to understand if there is more than a single aptamer for each target and discover if a sequence has been misused for several targets (as it was recently reported).
   2. Information about the functional utility in biologically relevant settings when applicable must also be included.
   3. The original report on which the aptamer was derived by SELEX must be mentioned when not obtained in the cited article to help the audience to trace it and contribute to reliability.
2. Most aptamers in this topic are obtained by Cell-SELEX, which is discussed in the text. However, an interesting variant called LIGs is not mentioned. A brief discussion about its pros and cons is needed.
3. It is said that in-silico SELEX can improve the success rate. This statement deserves further elaboration. Currently, aptamer modeling is based on software for RNA, though DNA aptamers are developed and Alphafold is still not reliable for this task as recently reported. Molecular simulations are not extended long enough (ns to 1 us) to ensure that macromolecules such as aptamers and proteins have a stable trajectory. Why should computational approaches be more successful than experimental ones? Discuss this. 
4. In ref 93 authors propose the possibility of turning an RNA aptamer into DNA in clear disagreement with accepted knowledge. A critical assessment and personal expert opinion of the authors is needed.
5. Modifications of aptamers are common in body fluid applications (LNA, pegylation, thioaptamers, etc), however, little is discussed on then. A general critical discussion is needed on the stability of RNA and DNA aptamers. Specific examples taken from table 2 should be discussed in more detail. Are these modifications really needed? Are they all intended to increase stability?
6. Section 4.3 is subdivided by transduction technique, but the last headline is not a transduction technique. I guess in forensic applications some analytical technique is used for verification/authentication.
7. The colorimetric approach based on AuNPs aggregation is mentioned and exemplified with a hybridization-based assay. However, the most common approach is the direct recognition by the wrapped aptamer that is “detached” from the NPs. It has been strongly demonstrated that this principle is misled. A comment on this issue should be valuable because most people can think about it.
8. The subsection devoted to electrochemical approaches only discuss different types of format assays but there is not a single example on this topic. If there are not examples this subsection should be removed. If it is maintained, the displacement assay needs discussion. The usual approach is the displacement of the aptamer or its complementary strand by the target. It is arguable that the labeled target has different affinity than the unlabeled target.

 

Please check whether:

1. the availability long term row is correct in table 1.
2. the target in ref 98 is CD30 as indicated in table 2
3. ref 109 and 124 are aimed at blood cells
4. all works where “cancer” was cited as the disease targeted are not hematological. If they are the specific disease should be mentioned as in the rest of works.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all the issues concerned. I think the manuscript can be accepted in the present form.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors has addressed carefully to reviewers' suggestions and comments, so the ms has been significantly improved in the quality and broad interest. The ms is suitable for publication.