g******c
发帖数: 18 | 1 To be honest, the so-called evidence of data manipulation or fabrication in
Han's Paper
is so weak.
So far, two major pieces of evidence have been presented.
The first one is the shape and migration of DNA bands seem not to be
similar
to those shown on protein SDS-PAGE. For these IDs, I will suggested a book
of DNA electrophoresis where you can read about some fundamental concepts
or
knowledge. The following book are helpful to understand the DNA
electrophoresis data. (DNA Electrophoresis, Edited by Svetlana Makovets,
Institute of Cell Biology,
University of Edinburgh, Edinburgh, UK. Published by Humann Press). The
visualization and capture process normally add non-linear effects on to the
band quantitation. One shall pays attention to these details when he/she
tries to interpret data of electrophoresis.
The second is the background noise issue on Han's picture, the compression
process will filter out the high frequency details of a picture, which
causes a "PS"-like consequence. However, a throughout noise evaluation of
the whole picture will tell the truth. If the noise level is consistent on
the whole picture, it's very unlikely the picture has been manipulated.
The "three tricks" of ssDNA transfection is not a new idea. It has been
hanging around for nearly 20 years. There are some literatures about it.
The injection or transfection efficiency of ssDNA seems to be the key.
Please make sure this step was done properly during a troubleshooting.
It's so sad to see that many young generation scientists are lack of
basic scientific or math knowledge. It's even more sad to see that they
are
listening to someone who scrutinizes a scientific paper on an unscientific
or
nonscientific way.
To be fair, to those who are questioning Dr. Han's paper, please write up a
scientific comment and submit to the journal. If your comments meet the
journal's scientific merit, your chance of publishing it will be very high.
Good luck to all. | n**********g
发帖数: 196 | 2 It's funny that you just picked the soft accusations to argue against.
Those are not "major concerns", especially PS. PS is intended to explain the
real major concerns, which are
1) lack of reproducibility
2) band shift pattern are conflicting with each other in Fig4a and 4e.
3) the integrated intensity of bands in Fig 4c is not consistent with their
size and molar ratio.
ssODN transfection tricks? No one has issue transfecting it with CAS9.
Take your time and read your book. Then come back with good answers. | n**********g
发帖数: 196 | 3 1) lack of reproducibility
二十多个实验室重复出来了。
--呵呵
2) band shift pattern are conflicting with each other in Fig4a and 4e.
电压不稳定，设备陈旧的。并不影响结论
－－呵呵
3) the integrated intensity of bands in Fig 4c is not consistent with their
size and molar ratio.
光线问题，相片数字压缩常见问题。并不影响结论
－－呵呵 | g******c
发帖数: 18 | 4 1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
it.
2) DNA band and migration are easily alternated by sample ionic strength and
buffer. The reason lies in the mechanism of DNA molecule electrophoresis is
so different from SDS-PAGE. DNA band shift is not a unusual result if DNA
sample denature or conformation is not consistent because DNA molecules
migrates on a electrical filed by the negative charge of its own phosphate
groups of which the charge is easily altered by buffer. However, protein
binds SDS molecules when it migrates on SDS-PAGE. SDS is a much effective
molecule to bestow negative charge to protein molecules and not easily
altered by buffer.
3) Visualization/staining and capture will add non-linear effects to DNA
quantitation. Un-even chemical staining and CCD translation is not always
guaranteed as good as a linear equation. A calibration curve is needed to
interpret the quantitation.
4) let's assume a 10% transfection may bring a good local substance
concentration working with CAS9, however, Dr. Han's enzyme is a total
different molecule which may
have different ssDNA concentration requirement.
Do you have a enzymatic constants regarding these two different enzymes at
37 oC? 2 times difference regarding kcat/Km will have huge impact on the
substance concentration requirement in order to achieve the same product
amount during the same time.
Please come back with more explanations on the scientific mechanism
the
their
【在 n**********g 的大作中提到】
: It's funny that you just picked the soft accusations to argue against.
: Those are not "major concerns", especially PS. PS is intended to explain the
: real major concerns, which are
: 1) lack of reproducibility
: 2) band shift pattern are conflicting with each other in Fig4a and 4e.
: 3) the integrated intensity of bands in Fig 4c is not consistent with their
: size and molar ratio.
: ssODN transfection tricks? No one has issue transfecting it with CAS9.
: Take your time and read your book. Then come back with good answers.
| d******t
发帖数: 27 | 5 图4里，Marker在笑的时候，样品却在哭，当离点样孔一样距离时。还是想不通。 | c********6
发帖数: 693 | 6 I was so sad that you are struggling for 50 cents. | n**********g
发帖数: 196 | 7 1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
it.
The biggest concern is not addressed by your answer.
2) DNA band and migration are easily alternated by sample ionic strength and
buffer.
No, I did not talk about band distortion. Read my question carefully. Fig 4a
and Fig 4e. No shift when cleavage sites are 30bp and then clear shift when
cleavage sites are closer.
3) Visualization/staining and capture will add non-linear effects to DNA
quantitation. Un-even chemical staining and CCD translation is not always
guaranteed as good as a linear equation. A calibration curve is needed to
interpret the quantitation.
If this is true, the entire paper needs to be trashed because all the
quantification and calculations relied on a linear relationship between
staining signal and DNA size/amount.
4) 10% transfection may bring a good local substance concentration working
with CAS9, however, Dr. Han's enzyme is a total different molecule which may
have different ssDNA concentration requirement.
No, no enzyme is involved. NgAgo DNA is transfected not NgAgo protein.
Read CAS9 work. | g******c
发帖数: 18 | 8 I was a nucleic acid research scientist, but not working on gene editing any
more
. The reason I am involved is to present some basic knowledge on nucleic
acid
research.
{1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
it.
The biggest concern is not addressed by your answer. }
Band shift sometimes can be seen due to sample ionic strength and buffer.
{ 2) DNA band and migration are easily alternated by sample ionic strength
and
buffer.
No, I did not talk about band distortion. Read my question carefully. Fig
4a
and Fig 4e. No shift when cleavage sites are 30bp and then clear shift when
cleavage sites are closer. }
Of course, in a real world, let's say in highly-regulated industry like
pharmaceutical industry, a calibration curve shall be submitted together
with the sample testing, however in academic it's not a practical way to do
so. Right now, so many electrophoresis results are presented on scientific
journals without a careful calibration. Dr. Han is not unique. I guess the
whole academic community does not have resource to do so and allow these
data published all the time.
{ 3) Visualization/staining and capture will add non-linear effects to DNA
quantitation. Un-even chemical staining and CCD translation is not always
guaranteed as good as a linear equation. A calibration curve is needed to
interpret the quantitation.
If this is true, the entire paper needs to be trashed because all the
quantification and calculations relied on a linear relationship between
staining signal and DNA size/amount. }
In living cells, these two technologies actually use enzyme to catalyze the
DNA
editing. Plasmid DNA is transfected into living cells to produce enzymes
that edit genomic DNA, so the enzymatic constants and substance (ssDNA)
concentration offered by transfection or injection both determine the
final product amount (editing efficiency).
It's chemistry in the end, not biology.
{ 4) 10% transfection may bring a good local substance concentration working
with CAS9, however, Dr. Han's enzyme is a total different molecule which
may
have different ssDNA concentration requirement.
No, no enzyme is involved. NgAgo DNA is transfected not NgAgo protein.
Read CAS9 work. }
and
4a
when
【在 n**********g 的大作中提到】
: 1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
: it.
: The biggest concern is not addressed by your answer.
: 2) DNA band and migration are easily alternated by sample ionic strength and
: buffer.
: No, I did not talk about band distortion. Read my question carefully. Fig 4a
: and Fig 4e. No shift when cleavage sites are 30bp and then clear shift when
: cleavage sites are closer.
: 3) Visualization/staining and capture will add non-linear effects to DNA
: quantitation. Un-even chemical staining and CCD translation is not always
| s******y
发帖数: 17729 | 9 纯好奇，Dr. chou卷进去背书的动机是啥，这个水很深啊，这时候跳入选边是不是有点
早。
in
book
【在 g******c 的大作中提到】
: To be honest, the so-called evidence of data manipulation or fabrication in
: Han's Paper
: is so weak.
: So far, two major pieces of evidence have been presented.
: The first one is the shape and migration of DNA bands seem not to be
: similar
: to those shown on protein SDS-PAGE. For these IDs, I will suggested a book
: of DNA electrophoresis where you can read about some fundamental concepts
: or
: knowledge. The following book are helpful to understand the DNA
| n**********g
发帖数: 196 | 10 So based on your reply,
1) still not addressed
2) still not addressed
3) clear your stance. Should Han paper be trashed because there is not
calibration curve, or should we trust the fields experience that such gels
should preserve their linear relationship between staining signal and DNA
amount?
4) you changed topic. You were talking about ""three tricks" of ssDNA
transfection". Stick to DNA transfection before we switch to what happens
after transfection.
any
reproduce
【在 g******c 的大作中提到】
: I was a nucleic acid research scientist, but not working on gene editing any
: more
: . The reason I am involved is to present some basic knowledge on nucleic
: acid
: research.
: {1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
: it.
: The biggest concern is not addressed by your answer. }
: Band shift sometimes can be seen due to sample ionic strength and buffer.
: { 2) DNA band and migration are easily alternated by sample ionic strength
| | | g******c
发帖数: 18 | 11 Dr. Chou is not a green hand. He is doing quite well right now and becoming
a rising star professor in SIBS.
I personally think Dr. Han's enzyme has relatively low activity of
catalyzing DNA compared to the CRISPR enzyme, which cause quite low success
rate of this experiment.
There is an improvement of timing and efficiency of ssDNA transfection after
plasmid DNA produces the enzyme in living cells.
I can feel the anxiety of some scientists questioning Dr. Han's work,
however the way they are questioning simply shows they do not have good
background on enzyme and nucleic acid chemistry.
【在 s******y 的大作中提到】
: 纯好奇，Dr. chou卷进去背书的动机是啥，这个水很深啊，这时候跳入选边是不是有点
: 早。
:
: in
: book
| g******c
发帖数: 18 | 12
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
This is a whole academic issue and how it is running right now. If the rule
changes, I guess the quality of scientific research will be improved.
However, who is going to pay this data validation process? It's not cheap if
the whole academic community decides to adapt the same rule that currently
used in industry.
The ssDNA transfection and injection efficiency determine the ssDNA
substance concentration in living cells. this concentration together with
enzyme catalytic constants determine the product amount and level in a given
time period. Do you understand the whole gene editing process?
The so called "tricks" are common sense for ssDNA transfection if you have
been doing nucleic acid research for a while. I simply am sharing my
knowledge. It's your decision to try them or not. If you have other ways to
improve ssDNA transfection efficiency, please go for it.
The Australian Scientist was using injection instead of transfection, which
is a physical way to bring ssDNA into living cell. The chemical infusion
process by transfection normally offers lower rate compared to the physical
way. This injection gives very high local concentration of ssDNA inside
living cells and so that higher level gene editing can be expected.
【在 n**********g 的大作中提到】
: So based on your reply,
: 1) still not addressed
: 2) still not addressed
: 3) clear your stance. Should Han paper be trashed because there is not
: calibration curve, or should we trust the fields experience that such gels
: should preserve their linear relationship between staining signal and DNA
: amount?
: 4) you changed topic. You were talking about ""three tricks" of ssDNA
: transfection". Stick to DNA transfection before we switch to what happens
: after transfection.
| n**********g
发帖数: 196 | 13 1) Do you understand statistics?
2) you need to address why they conflict with each other. (Fig 4a and 4e).
You need to read others’ question first.
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
3) you need to be specific to Han’s paper.
This is a whole academic issue and how it is running right now. If the rule
changes, I guess the quality of scientific research will be improved.
However, who is going to pay this data validation process? It's not cheap if
the whole academic community decides to adopt the same rule that currently
used in industry.
4) “This injection gives very high local concentration of ssDNA inside
living cells and so that higher level gene editing can be expected.”——
Your theory of "local concentration" completely makes no sense. Do you
understand the whole gene editing process?
Do you understand how long does it take to synthesize a protein from
transfected plasmid? Do you understand a word called “diffusion rate”?
The ssDNA transfection and injection efficiency determine the ssDNA
substance concentration in living cells. this concentration together with
enzyme catalytic constants determine the product amount and level in a given
time period. Do you understand the whole gene editing process?
The so called "tricks" are common sense for ssDNA transfection if you have
been doing nucleic acid research for a while. I simply am sharing my
knowledge. It's your decision to try them or not. If you have other ways to
improve ssDNA transfection efficiency, please go for it.
The Australian Scientist was using injection instead of transfection, which
is a physical way to bring ssDNA into living cell. The chemical infusion
process by transfection normally offers lower rate compared to the physical
way. This injection gives very high local concentration of ssDNA inside
living cells and so that higher level gene editing can be expected. | g******c
发帖数: 18 | 14 When will your statistic data collection be completed? Let's talk about the
success
rate then.
Your comments are so interesting. If you do not know the science, just be
patient and wait for more answers.
{1) Do you understand statistics?}
-----------------------------------
Ok, alright. I spent some time to explain it to you.
Fig 4a, the band distance from 1000 to 250 is 2.5 cm on my monitor. Based on
linear regression of Log(size) and migration distance, 30 nt will give out
1.3 mm difference around the position of 500 on this picture, however the
band width diffuses to 1.5 (
G6 lane) and 2.0 mm (G13 lane). Do you think 1.3 mm difference (resolution)
can be achieved on peaks with a width of 1.5-2 mm? More sophisticated
evidence shall be present if you have.
Fig 4e, the same calculation will be applied in addition to difference
sample preparation.
{2) you need to address why they conflict with each other. (Fig 4a and 4e).
You need to read others’ question first. }
---------------------------------------
If the journal adapts the industry rule, I will suggest everyone obeys the
rule and reject any paper not in compliance with the rule . Most labs
publish electrophoresis data without a calibration/validation process. These
to-be-retracted paper may included yours as well if you are doing CRISPR. I
am specifically talking whether Dr. Han's is in compliance with the current
rule or not.
{3) you need to be specific to Han’s paper. }
-----------------------------------------
Local concentration means the concentration around this editing enzyme.
I assume that you trying to mention the timing of enzyme translated and co-
localized with ssDNA by asking me the protein translation and diffusion rate
. I accept your questions and try to answer them now.
You are not the first one to mention the synchronization and co-localization
of enzyme and substances in living cells. I have mentioned this issue on
another post in this tread. Timing to feed ssDNA substance to the enzyme can
be improved in Dr Han's system. The more synchronized the ssDNA molecules
sit with the enzyme molecules, the better the catalytic reaction efficiency
will achieved.
Do you have other questions regarding the DNA editing mechanism?
{ 4) “This injection gives very high local concentration of ssDNA inside
living cells and so that higher level gene editing can be expected.”——
Your theory of "local concentration" completely makes no sense. Do you
understand the whole gene editing process?
Do you understand how long does it take to synthesize a protein from
transfected plasmid? Do you understand a word called “diffusion rate”? } | n**********g
发帖数: 196 | 15 1) The current low (zero) verified success rate is sufficient for people to
question the story.
When will your statistic data collection be completed? Let's talk about the
success
rate then.
Your comments are so interesting. If you do not know the science, just be
patient and wait for more answers.
2) “Do you think 1.3 mm difference (resolution)
can be achieved on peaks with a width of 1.5-2 mm?” The answer is yes
because of Fig 4e. Your statement is “difference
sample preparation” is completely wrong. They described their protocol of
running gels in supplement Page 15.
“Mix the 10 μl T7EI reaction product with 2 μl 6x loding buffer (Biolab)
and ran electrophoresis at 140v for 30min. ”
You cannot make up an excuse that are different from what the authors
claimed and expect people to buy it.
Fig 4a, the band distance from 1000 to 250 is 2.5 cm on my monitor. Based on
linear regression of Log(size) and migration distance, 30 nt will give out
1.3 mm difference around the position of 500 on this picture, however the
band width diffuses to 1.5 (
G6 lane) and 2.0 mm (G13 lane). Do you think 1.3 mm difference (resolution)
can be achieved on peaks with a width of 1.5-2 mm? More sophisticated
evidence shall be present if you have.
Fig 4e, the same calculation will be applied in addition to difference
sample preparation.
3) You are avoiding my question. Answer directly. Should Han paper be
trashed because there is not
If the journal adapts the industry rule, I will suggest everyone obeys the
rule and reject any paper not in compliance with the rule . Most labs
publish electrophoresis data without a calibration/validation process. These
to-be-retracted paper may included yours as well if you are doing CRISPR. I
am specifically talking whether Dr. Han's is in compliance with the current
rule or not.
4) You don’t even understand what yourself are talking about. The opposite
word of “local” is “global”. ssDNA is freely diffused in cells and there
is no “local concentration around this editing enzyme”, unless you
discover a mechanism for local enrichment of ssDNA around NgAgo.
Your theory of “synchronization” remains to be tested. But theoretically,
it makes little sense because new NgAgo protein is constantly synthesized in
the presence of ssODN from a few hours after transfection to a few days.
Maybe delaying transfection of ssODN by a few hours will avoid potential
degradation of ssODN during those few hours. Han clearly showed that he
could achieve better editing by just co-transfecting NgAgo construct and
ssODN. So your theory of “better synchronization” cannot be used to
explain other’s failure to reproduce his result.
Do you have other questions regarding the DNA editing mechanism?
Local concentration means the concentration around this editing enzyme.
I assume that you trying to mention the timing of enzyme translated and co-
localized with ssDNA by asking me the protein translation and diffusion rate
. I accept your questions and try to answer them now.
You are not the first one to mention the synchronization and co-localization
of enzyme and substances in living cells. I have mentioned this issue on
another post in this tread. Timing to feed ssDNA substance to the enzyme can
be improved in Dr Han's system. The more synchronized the ssDNA molecules
sit with the enzyme molecules, the better the catalytic reaction efficiency
will achieved.
Do you have other questions regarding the DNA editing mechanism?
{ 4) “This injection gives very high local concentration of ssDNA inside
living cells and so that higher level gene editing can be expected.”——
Your theory of "local concentration" completely makes no sense. Do you
understand the whole gene editing process?
Do you understand how long does it take to synthesize a protein from
transfected plasmid? Do you understand a word called “diffusion rate”? } | g******c
发帖数: 18 | 16 Before I start, I’d like to make my point crystal-clear. This will be
serving as my
last post on this subject and I am not interested in any college-level
discussion.
How soon do you want to close your statistical data collection? I guess
this time window of reproducibility will be determined by the journal and
the whole scientific community. Dr. Han’s fate does not concern me from
the beginning. However, I do care the quality of arguments against Dr. Han’
s
paper written up by some green-hands. The weaker statement against Dr. Han
’s paper is, the more ridiculous the whole discussion looks like.
My calculation is based on DNA migration theory and electrophoresis common
sense. Peak tailing or fronting, diffusion causes bad resolution on
electrograms. What’s your calculation result on the resolution for the
figure 4a? The reason I pointed this out is the argument against Dr. Han’s
paper looks so lack of science.
I believe in terms of Calibration/validation requirement, Dr. Han did not
violate the current rule used by the whole academic community. No violation
means no violation. No violation leads to no punishment. It leave
individuals to decide to believe whatever they believe, however the public
rule shall not be voided just simply to one specific paper or person. This
is called “rule of law”. I have explained this so clearly and please be
professional and no more chasing on this one.
OK, since you have started the conversation, let me elaborate the whole
editing catalytic process of genome editing (not the plasmid DNA editing)
for you. When the enzyme translated in the cytosol, it needs to be
transported by nuclear localization sequence into a cell nucleus to function
as an editing enzyme on genome DNA. ssDNA shall be brought by some
biological mechanism or by simple diffusion that is very unlikely in living
cells. My term “local concentration” just to emphasize the effective
concentration of 5’p-ssDNA in nuclei that is the real concentration the
editing reaction will be governed. What’s wrong with this term of “local
concentration”? The expression level of the enzyme shall be elaborated to
be more specific to the concentration in nuclei, not on the whole cell level
. The nuclear transportation system has a limit capacity per cell, which
allows a certain amount of the enzyme molecules to enter into cell nucleus.
The timing and co-localization of ssDNA and enzyme in nuclei is the key to
the editing rate. Of course, the conformation of the target genomic DNA
sequence functions as another key factor to the rate. This whole chemical
editing reaction yield can be improved by tuning every steps of transfection
/injection, cell proliferation cycle, NLS, ssDNA quality, timing, the enzyme
catalytic nature. Every steps determine the outcome of gene editing
production amount.
The CRISPR system has been improved a lot from the beginning by optimizing a
lot factors that affects the gene editing reaction rate. It’s not just a
tool for biologists, it’s chemistry in the end of day.
I seriously suggest those scientist questioning Dr. Han’s paper to write up
a negative comment and submit to the journal, so that the discussion will
be more scientific and the current chaos will be ending soon. | m******e
发帖数: 459 | | n**********g
发帖数: 196 | 18 You comments are funny and clearly show you don't understand gene editing or
this paper at all. The reason you found evidences against Han are weak is
simply because you picked weak ones to fight against.
1) You avoided to answer the lack of reproducibility. You want to "window"
as long as possible to deny this concern. But Han distributed plasmids to
many Chinese laboratories during talks and more than 100 labs acquired his
NgAgo plasmid through Addgene. Yet there is only a single (unverified) case
that claimed to detect weak genome editing (<5%). Existing data is enough to
draw a conclusion.
2) You avoided to answer the conflict between Fig 4a and Fig 4e. You
intentionally picked Fig 4a to answer but it is NOT the question. How
ridiculous it is!
3) About "calibration/validation requirement"--when I questioned you why
smaller bands had higher integrated intensity than larger bands in Fig 4c,
YOU said Han's quantification of bands was problematic because staining
signal was not in linear relationship. YOU actually trashed his paper by
this accusation even though you don't want to admit it. BTW, the field
believes a linear relationship and that's why his result (Fig4c) is so weird
.
4) Your new statement of "local concentration" clearly shows you DON"T
understand how NgAgo works. NgAgo is synthesized in the cytoplasm and it
needs to be loaded with ssODN immediately after/during it is synthesized.
Han clearly showed that mixing pre-made NgAgo with soODN at 37 celsius
cannot assemble a complex. So your theory of assembly NgAgo/ssODN in the
nucleus is completely BASELESS! Your timing theory just shows that you don't
even understand this paper.
And finally, do you really think we need your advice on how to comment peer'
s work in a scientific way? So naive if you do think so, and I can guarantee
you are wrong. What needs to be done is already done. | m****a
发帖数: 270 | 19 我们不能因为别人是“新手上路”就质疑别人的专业水平，也不能因为看过《Э-505型
挖掘机
维修指南》就说自己是老司机一样。。。
【在 m******e 的大作中提到】
: 新手上路，again。。。
| g******c
发帖数: 18 | 20 You leave me no choice but . I can not stand someone without any chemistry
sense when he/she is a biologist.
Chemical reaction is a on/off equilibrium. Some enzymes pick up the first
substance first and then the second one if the space arrangement is allowed
in the active site; some enzymes bind to a coordinated substance complex if
the reaction needs both substances available at the same time to fit into
the active site. It depends on the nature of individual enzymes. Since ssDNA
can not bind to the editing enzyme alone in vitro, the conclusion will be
the enzyme needs the second substance that is a conformation-ready genomic
DNA to form a functional enzyme-ssDNA-genomic target DNA complex to complete
the reaction.
In addition, Let's assume the enzyme will bind ssDNA in cytotol as in your
statement. As known to all, any chemical binding between two molecules goes
through a equilibrium process. There is an on and an off rate between them.
Enzyme normally does not have a very high affinity to its substance, which
is how the enzyme can catalyze the substances and let substance leave its
active site. In most of case, the Km value ranges from 1-100 mM in vitro.
There is no way that the enzyme-ssDNA complex will last forever till the
complex is transported into the nuclei by NLS and complete the reaction.
During the transportation, there is always an on/off equilibrium. If the
effective (local) concentration of ssDNA inside nuclei is too low, let's say
below its Km, the off rate governs the equilibrium and the reaction yield
will be really low.
Let's look at the 3D structure the enzyme in PDB. If the enzyme-ssDNA
complex shall be preserved before the reaction starts, the genomic DNA will
be prevented from accessing the active site by the steric effects caused by
ssDNA bound in the active site.
Chemistry common sense leads the way of biologists to a success.
Savvy?
or
case
to
【在 n**********g 的大作中提到】
: You comments are funny and clearly show you don't understand gene editing or
: this paper at all. The reason you found evidences against Han are weak is
: simply because you picked weak ones to fight against.
: 1) You avoided to answer the lack of reproducibility. You want to "window"
: as long as possible to deny this concern. But Han distributed plasmids to
: many Chinese laboratories during talks and more than 100 labs acquired his
: NgAgo plasmid through Addgene. Yet there is only a single (unverified) case
: that claimed to detect weak genome editing (<5%). Existing data is enough to
: draw a conclusion.
: 2) You avoided to answer the conflict between Fig 4a and Fig 4e. You
| | | n**********g
发帖数: 196 | 21 hey, dude. I beg you--read the paper before replying to me. What you said
just contradict to Han's paper. I had to laugh for a while before I can
type. Let me finish dinner first. | m****a
发帖数: 270 | 22 连韩春雨的论文都没看懂在这儿瞎逼逼，还 enzyme-ssDNA，there is always an on/
off equilibrium.
麻烦你去看看论文原文和supplemental figure 1
NgAgo is faithful to its original guide and does not allow DNA swapping at
37 °C (Fig. 2d), similar to mammalian Ago2, which cannot exchange its bound
oligos with free oligos at 37 °C. This feature of NgAgo minimizes the
possibility that it will be loaded with unexpected ‘guides’.
。。。。NgAgo follows a ‘one-guide-faithful’ rule, that is, a guide can
only be loaded when NgAgo protein is in the process of expression, and, once
loaded, NgAgo cannot swap its gDNA with other free ssDNA at 37 °C.
你要是学有余力再去看看2012 年的cell paper, 关于Ago的结构研究。
http://www.sciencedirect.com/science/article/pii/S0092867412006186
RNA 和 Ago结合后会非常稳定的complex，连蛋白酶都降解不了。
allowed
if
ssDNA
complete
【在 g******c 的大作中提到】
: You leave me no choice but . I can not stand someone without any chemistry
: sense when he/she is a biologist.
: Chemical reaction is a on/off equilibrium. Some enzymes pick up the first
: substance first and then the second one if the space arrangement is allowed
: in the active site; some enzymes bind to a coordinated substance complex if
: the reaction needs both substances available at the same time to fit into
: the active site. It depends on the nature of individual enzymes. Since ssDNA
: can not bind to the editing enzyme alone in vitro, the conclusion will be
: the enzyme needs the second substance that is a conformation-ready genomic
: DNA to form a functional enzyme-ssDNA-genomic target DNA complex to complete
| n*u
发帖数: 373 | 23 是Dr Qiu,不是Dr Chou
你们小学语体教吗 | g******c
发帖数: 18 | 24 No matter how the kon/koff ratio favors the complex formation, a non-
covalent binding will definitely go through a on/off equilibrium. The
Antibody-
antigen interaction may be the highest affinity (0.1-1 nM of Kd) complex
that people have ever studied in biological systems, however the equilibrium
does exist in the Ab-
Ag complex. That's how SPR analysis is working to assess the affinity.
I do not believe that non-covalent complex will last forever when the
effective
concentration of substance decreases to very low level, let's say 10% of Kd
(or Km). It's simply against the chemical rule if non-covalent interaction
does not obey the same equilibrium rule.
In addition, an in vitro assessment of the enzyme assay will be helpful to
understand its activity, Km, kcat. Or you can tell me what the Kd of NgAgo
is and then we can talk about the equilibrium constant.
Protease can not digest the protein complex you mentioned is due to the
conformation change induced by the substance binding that happens when the
substance concentration favors the complex formation. A very low affinity
substance (ligand) also can protect a bound protein from protease digestion
as long as it's available in the system all the time at a very high
concentration let's say 10x Kd.
Again, your comments needs to be more elaborated and more quantitatively. I
do not like qualitative description of a chemical reaction.
bound
once
【在 m****a 的大作中提到】
: 连韩春雨的论文都没看懂在这儿瞎逼逼，还 enzyme-ssDNA，there is always an on/
: off equilibrium.
: 麻烦你去看看论文原文和supplemental figure 1
: NgAgo is faithful to its original guide and does not allow DNA swapping at
: 37 °C (Fig. 2d), similar to mammalian Ago2, which cannot exchange its bound
: oligos with free oligos at 37 °C. This feature of NgAgo minimizes the
: possibility that it will be loaded with unexpected ‘guides’.
: 。。。。NgAgo follows a ‘one-guide-faithful’ rule, that is, a guide can
: only be loaded when NgAgo protein is in the process of expression, and, once
: loaded, NgAgo cannot swap its gDNA with other free ssDNA at 37 °C.
| S**********e
发帖数: 620 | 25 专业人士过招，这个帖子比较好看点
don't stop, keep on going | g******c
发帖数: 18 | 26 From the beginning, Dr. Han's fate does not concern me. He may not disclose
all details or hide his true work. I simply want to share my
work experience and post my points here.
The confusion on Dr. Han's work can be clarified when time goes by. There is
no solid yes or no -
evidence against him right now. If you have, please write it up and submit
to the editor. In this way, Dr. Han will have to come back with some solid
evidence.
However, some comments on Urea-PAGE pictures have been posted and twittered
around. Background noise caused by compression was mistaken as a "PS" issue;
The shape of DNA bands caused by imperfect electrophoresis experiments is
twittered as a piece of evidence against the paper; Assessment of resolution
without careful calculation was made and used
against the paper. These comments are so weak and without scientific
merits. I do not care about the outcome of this paper. However, what I
emphasize is always that quality of arguments.
Please write up a comment and submit your arguments as soon as possible,
which is the best way to push Dr. Han to defend his work. If he still has
the samples, he urgently needs good electrophoresis equipment to reproduce
the PAGE work in his paper.
【在 n**********g 的大作中提到】
: hey, dude. I beg you--read the paper before replying to me. What you said
: just contradict to Han's paper. I had to laugh for a while before I can
: type. Let me finish dinner first.
| n**********g
发帖数: 196 | 27 OK, you want basic chemistry and here is basic chemistry.
“Since ssDNA
can not bind to the editing enzyme alone in vitro, the conclusion will be
the enzyme needs the second substance that is a conformation-ready genomic
DNA to form a functional enzyme-ssDNA-genomic target DNA complex to complete
the reaction. ”
----This is so wrong. There could be 100 reasons why an enzyme does not bind
a substance. For example, temperature, pH. Most importantly, it directly
contradicts Han’s data (Fig 1,2).
“As known to all, any chemical binding between two molecules goes
through a equilibrium process. There is an on and an off rate between them.
During the transportation, there is always an on/off equilibrium. If the
effective (local) concentration of ssDNA inside nuclei is too low, let's say
below its Km, the off rate governs the equilibrium and the reaction yield
will be really low. ”
----Okay, you know on/off rate! Of course you need to take concentration and
on/off rate into consideration. But without any data, you assumed that
lipid-based transfection introduces too low concentration of ssODN to
promote the forward reaction and you used this to explain other people’s
failure. This is completely baseless. Usually you use less ssODN if you do
physical injection, so the effective concentration of ssODN is not
necessarily much higher than lipid-based transfection. In fact, Han used
lipid and he got really good result, which indicates the effective
concentrations of those components are well above Kd. So you are using your
imagination to argue against people’s data.
“Let's look at the 3D structure the enzyme in PDB. If the enzyme-ssDNA
complex shall be preserved before the reaction starts, the genomic DNA will
be prevented from accessing the active site by the steric effects caused by
ssDNA bound in the active site.”
--Sure you can do this practice if you solve the structure of NgAgo. But you
won’t be able to make any conclusions no matter what your finding is.
First, ssODN may not bind to the active site (by active site, I assume you
mean the cleavage site). Second, you can have another 100 components (
enzymes, ATPs) to help relieve any steric interference. So it is perfectly
possible to have steric interference in preassembled enzyme/substance and
still have the reaction going forward. You need to improve your logic and
your experimental design.
In fact, Han found that you cannot get the reaction going forward when you
incubate Ago, ssODN and target (Fig 3c) and you have to use pre-assembled
Ago/ssODN in order to cleave target. Pre-assembled NgAgo/ssODN is actually
favored. So your theory of high local concentrations of those three
components making it assemble and cleave makes no sense at all. It is more
likely the NgAgo loading is the rate limiting step and it needs either of
the two conditions 1) high temperature to overcome the energy barrier or 2)
a certain conformation that shortly appear after protein translation or
during translation. The pre-assembled NgAgo/ssODN does not seem to have
steric interference otherwise it won’t be able to cleave target.
allowed
if
ssDNA
complete
【在 g******c 的大作中提到】
: You leave me no choice but . I can not stand someone without any chemistry
: sense when he/she is a biologist.
: Chemical reaction is a on/off equilibrium. Some enzymes pick up the first
: substance first and then the second one if the space arrangement is allowed
: in the active site; some enzymes bind to a coordinated substance complex if
: the reaction needs both substances available at the same time to fit into
: the active site. It depends on the nature of individual enzymes. Since ssDNA
: can not bind to the editing enzyme alone in vitro, the conclusion will be
: the enzyme needs the second substance that is a conformation-ready genomic
: DNA to form a functional enzyme-ssDNA-genomic target DNA complex to complete
|
|
