Dual Catalytic Hairpin Assembly-Based Automatic Molecule Machine for Amplified Detection of Auxin Response Factor-Targeted MicroRNA-160

Round 1

Reviewer 1 Report

The manuscript describes a type of catalyzed hairpin assembly (CHA) reaction for the fluorescence detection of miRNA160. The unique feature is the dual-catalytic cycle CHA which involves the formation of  4WJ product and concomitant regeneration of the miRNA trigger. While the signal increase is modest (around 4-fold) and the kinetics is rather slow (requires 8 h to complete), it was possible to detect the target down to 10 pM with good specificity. Publication can be recommended subject to the following issues being appropriately addressed:
major points
- Please expand the calibration graph (Fig 4) in the range of 0-1 nM.
- The formation of the 4WJ should be independently verified (by gel electrophoresis or other means).
- The testing was performed only with miRNA60D as a surrogate of miRNA60. To claim the detection of miRNA160, the real RNA target must be tested. 
- Please indicate whether the "blind sample test" (Table S2) was done with synthetic oligonucleotide target or real sample? If done with synthetic oligos, please indicate whether it is the real miRNA160D or miRNA160 (the latter is highly preferred), and please make sure that the sample matrix is relevant to the real sample testing (i.e., results in the pure buffer should not be claimed for applicability in real samples)
presentational points
- What are the exact HP2 and HP3 being used in Fig 2? There are several of them (HP2-I to VI, HP3-I to V) being used in the paper.
- The kinetics in Fig S1 was plotted on the wrong x-axis scale. It is neither a linear nor a logarithmic scale. Please correct this.
- There are several grammatical mistakes such as "..it has no the combined.." in p4 line 155. Please carefully proofread again. 

Author Response

We have carefully read the letter and are pleased to hear the positive feedback. We have revised our manuscript according to the valuable comments. All the changes made are clearly indicated with yellow background in Supporting Information for Review Only and the one-by-one responses are attacted.

Author Response File: Author Response.pdf

Reviewer 2 Report

Tremendous achievements have been made regarding CHA based bioanalysis from origin to widespread application. Most of the CHA systems have been tuned in to detect a variety of DNA. Thus, the article by Lei Wang et al. makes a certain contribution to solving the urgent problem of bioorganic chemistry.

1) CHA method itself is known for years, moreover there are a number
of its modifications applied for different targets including miRNAs.
The authors should explain why they choose exactly this scheme with
dual CHA and compare it with other existing schemes.
2) I see no solid reason to name this technique as "automatic molecule
machine (AMM)" because in scientific practice this terminology is used
mainly for particles which make some movements and have some energy,
but nor just self-amplifying systems. Otherwise we should name it as
AMM, even HCR Northern blotting.
3) M&M section and further results sections are confusing when
describing target oligos. The authors claim they detect miRNAs however
they used for all targets only their DNA oligonucleotide analogs. This
is explained in section 2.2. However further the authors used both
names either miRNA160  or miRNA160ВD thus it could be interpreted
wrongly.
4) The same criticism for section 2.5. The authors wrote: "the common
plant miRNAs 200 (miRNA156, miRNA159, miRNA164, miRNA390, miRNA396) were detected against 201 miRNA160 under identical conditions." However, we see on the figure 5 legend that they used DNA analogs only.
5) Thus, an essential real RNA control is needed for the study to
compare sensitivity of the method in case of RNA and DNA
6) At this stage of the research the suggested technique is applied
only for in vitro experiments. However, the authors describe in
paragraph 2 in the introduction section difficulties in the detection
of microRNA, but all these cases are actual for in vivo. So if the
general idea of the study was to establish a new method for sensitive
detection of miRNA190, this should be verified at the end of the
article, preferably with some total RNA extracts (probably
miRNA-enriched) from some plants.

7) “The reaction continues autonomously until most 114 DNA probes are assembled into F-DJ” and also Figure S1. If this mechanism is correct, then it is equivalent to a branched chain reaction, in which both the initial miR160 and the reaction product act as an initiator of a new chain. To prove this mechanism, it is necessary to investigate the dependence of the rate of accumulation of the fluorescent product on the concentration of miR160 at constant concentrations of all HP. The data given in the supplementary is not enough.

8) Line 104. May be it must be detection but not detention. 

9) In Ref. 47 there are only initials and no surnames.

Author Response

We have carefully read the letter and are pleased to hear the positive feedback. We have revised our manuscript according to the valuable comments. All the changes made are clearly indicated with yellow background in Supporting Information for Review Only and the one-by-one responses are attacted.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript ‘Dual Catalytic Hairpin Assembly-based Automatic Molecule 2 Machine for Amplified Detection of Auxin Response Fac-3 tor-targeted MicroRNA-160’ is devoted to the development of the Automatic Molecule Machine (AMM) for sensitive detection of microRNA160 in plants. The developed approach provides accurate and selective detection of miRNA160. The results are of high interest due to the significant difficulties in miRNA160 detection by existing methods. The introduced biosensor can be further applied for the detection of other plant miRNAs, all sharing the same demanding properties, such as small size, low expression levels and high sequence homology.

There is few minor comments on the manuscript:

1. Figure 1 has small shapes with low-contrast colors, and thus is hard to comprehend.

2. There are no spaces before the square brackets of the references in the text.

3. Line 66 - reliable

Author Response

We have carefully read the letter and are pleased to hear the positive feedback. We have revised our manuscript according to the valuable comments. All the changes made are clearly indicated with yellow background in Supporting Information for Review Only and the one-by-one responses are attacted.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I am mostly satisfied with the authors' correction. There are still some suggested minor changes before it can be accepted for publication:
line 29: achieving the detection limit as low as 10 pM for miRNA160 -> add "as deduced from its corresponding DNA surrogates" after miRNA160.
line 213: the common plant miRNAs -> the DNA surrogates of common plant miRNAs
The sequence of the synthetic RNA target referred to in Fig S4 and line 201 should be provided in Table S1.

Author Response

Thank you for your comments. We have provided a point-by-point response to the comments and upload as a word file. Please see the attachment.

Author Response File: Author Response.pdf