[RASMB] AUC measurements in CsCl?

Thu Jun 6 18:26:39 PDT 2019

Dear Peter,

Thank you for your paper.  It is useful to know that it is possible to measure species of this size using direct boundary approaches, also useful and interesting that a high dilution is necessary to avoid electrostatic effects – this is probably because of the charge on the nucleic acid? 

I wonder if at this dilution, 0.005%wt/v, if there would be sufficient signal for a 10MDa species to use the absorbance optics.  I would assume so since nucleic acids absorb strongly at 260nm, but my assumptions oft go awry.  Perhaps another reason to go down the CsCl road.

Best regards,

John Sumida

Molecular Analysis Facility

University of Washington

From: Prevelige, Peter Edward, Jr [mailto:prevelig at uab.edu] 
Sent: Thursday, June 06, 2019 5:58 PM
To: John Sumida <jpsumida at uw.edu>
Cc: Borries Demeler <demeler at gmail.com>; RASMB <rasmb at list.rasmb.org>
Subject: Re: [RASMB] AUC measurements in CsCl?

Maybe worth noting that it was important in the M&S experiment that the DNA was sheared.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1307605/

Sent from my iPad

On Jun 6, 2019, at 7:44 PM, John Sumida <jpsumida at uw.edu <mailto:jpsumida at uw.edu> > wrote:

Dear Borries,

Thank you for your response which is 100% on the mark with regards to the
expected deliverables.  

While the work being proposed is not an exact repeat of the Meselson & Stahl
experiment (one of my favorites as well) it is along the same vein.  The
work is attempting to use the methods outlined in the Flory Vinograd work
(which I've attached) which uses bromodeoxyuridine as a mass label (an
interesting idea ) instead of N15; and yes, my collaborators are seeking to
know the relative quantity of the species detected and the sedimentation
value is less important.  

Interesting also that in the Messelson and Stahl experiment they used whole
cell lysate, this was not significant to me when I read this paper back in
the day, but now this seems particularly useful?

I do not have the 3mm cells here, do you think this experiment is still
doable in a standard two channel SV cell?

Just so I am clear on the reason to use CsCl in this particular case is the
ability to detect small amounts of DNA, the large masses involved, which
would require a long slow speed spin, and avoid complications from
heterogeneity that may be existant at a given buoyant density and which may
be difficult to interpret?

Thanks so much for this information, I am eager to get started.

Best regards,

John Sumida
Molecular Analysis Facility
University of Washington

In many spheres of human endeavor, from science to business to education to
economic policy, good decisions depend on good measurement.
Ben Bernanke

-----Original Message-----
From: Borries Demeler [mailto:demeler at gmail.com] 
Sent: Thursday, June 06, 2019 4:36 PM
To: John Sumida <jpsumida at uw.edu <mailto:jpsumida at uw.edu> >
Cc: RASMB <rasmb at list.rasmb.org <mailto:rasmb at list.rasmb.org> >
Subject: Re: [RASMB] AUC measurements in CsCl?

Hi John,
Accurate molar mass measurement of any solute by SV or SE methods requires
an accurate vbar. This varies depending on ionic strength, ie.
hydration - so, do you know that? And if so, how? Cesium will bind to the
DNA changing its vbar (it is a very dense molecule). SE will also be
challenging since equilibrium will take forever to be achieved at lower
speed, and the steep gradient you are likely going to see at the bottom of
the cell will be distorted by refractive effects and not give you a lot of
points. Moreover it would be nearly impossible to resolve any heterogeneity
in the samples from size. So, I wouldn't use SE.

For SV experiments to additionally get a molar mass you need to be able to
measure the diffusion coefficient accurately. Large molecules like this have
very little diffusion signal. You would have to run for a very long time at
a very slow speed to get sufficient accuracy.

Everything else requires making assumptions and estimates. So I think your
traditional approaches for SV and SE will not be satisfactory.

It sounds like your collaborators are asking for the classic experiment
performed by Meselson & Stahl in 1958 (paper attached, one of my favorites,
I make my students read it). The different amounts of incorporated label
impart slightly different molar masses that can be separated based on the
different densities using buoyant density equilibrium gradients. It is the
CsCl gradient that will make the two populations buoyant at different places
in the cell. As you can see in the paper, this works quite well, and even
better with modern equipment.

For this to work well in an AUC, I recommend using 3 mm centerpieces.
It minimizes the CsCl gradient signal and hence attenuates the refractive
effect from the gradient. Use optically pure CsCl so you don't have
background at 260 nm where the DNA will absorb. This *should* work, though I
haven't tried it. While it will not give you accurate molar masses it should
resolve the two populations of labeled and unlabeled DNA. As an added bonus,
any heterogeneity from shearing is pretty much masked, since all molecules
will go where they have the same density, which differs for labeled and
non-labeled molecules. Speeds between
40-50 krpm should work well. Let us know how it works out. You won't need a
lot of DNA to make this measurement, since all the DNA will migrate to a
small spot and concentrate there to make a big peak.
Follow the paper to get the CsCl concentration correct. This is important
and needs to be matched to the DNA samples so you get the best separation.

In the end you still don't know absolute molar masses but I don't think you
actually need that. I bet your collaborators only care about the relative
amount of each population. You could also try this by standard velocity
analysis, but there the result will also encounter concentration dependent
non-ideality, but may resolve the boundaries from the two different
densities. Heterogeneity may be reducing your confidence here.
Meselson and Stahl, though an old paper, still seems like the most elegant
way to solve this problem. Let us know how it works out.

Good luck!

-Borries

Thu, Jun 06, 2019 at 02:49:44PM -0700, John Sumida wrote:

Dear RASMB,

First I have to thank all those who curate this list server as over 

the last

10 years as a manager of the lab I run, it has and continues to prove 

a hugely valuable resource for me and the people I serve.

My question today has to do with the analysis of DNA and it stems from 

a user interest in performing an AUC equilibrium experiment in the 

presence of CsCl.  My experience to date has been with nanomaterials 

and proteins so DNA is a new material for me, however I am under the 

impression that running AUC samples in presence of CsCl is 

problematical because of the gradient CsCl itself will generate.

The estimated mass of the species of interest is in the range of 10 

mega Daltons; this is also a size regime that is new to me.

The objective of the experiment is to characterize nucleic acids 

extracted from cells which will have a label increasing the mass of 

the nucleic acid depending on the number of times it has been replicated.

Two questions:

Is CsCl necessary to determine the size distribution of molecular 

species

10MDa?

Can a CsCl experiment be performed using equilibrium measurements?

Thank you for considering my question.

Best regards,

John Sumida

Molecular Analysis Facility

University of Washington

In many spheres of human endeavor, from science to business to 

education to economic policy, good decisions depend on good measurement.

Ben Bernanke

_______________________________________________

RASMB mailing list

RASMB at list.rasmb.org <mailto:RASMB at list.rasmb.org> 

http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

<Flory Vinograd BrdU mtDNA 1973.pdf>

_______________________________________________
RASMB mailing list
RASMB at list.rasmb.org <mailto:RASMB at list.rasmb.org> 
http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20190606/5380767b/attachment-0002.html>