RE: 5'-Thiol on oligos

From: Singleton, David H (david_h_singleton@groton.pfizer.com)
Date: Thu Mar 23 2000 - 08:16:48 EST

Winnell:
IMHO from a peptide chemist:

To tell / quantitate useable S-H thiols, spectrophotometric DTNB is best.
(G. L. Ellman, Arch. Biochem Biophys. 82,70 (1989); other peptide literature
awash with references). This will give you a read out for the totality of
disulfide formed (DTNB will be negative) and for complete reduction to S-H
free thiols (DTNB positive and quantitative for umol SH expected).

One Caveat: With excess DTT from reduction, this will not work. DTNB will
react with any and all free S-H. You must isolate by HPLC, or other
techniques (Dialysis has worked well; I don't know with oligonucleotides) to
look at only your product of interest.

You're right; the 3 species should elute differently by HPLC. If this is
true, that's your easiest readout for oxidation state.

David H. Singleton
Scientist
Pfizer Central Research
PO Box 8118-101
Eastern Point Road
Groton, CT 06340

-----Original Message-----
From: Winnell H. Newman [mailto:winnell@ncsu.edu]
Sent: Wednesday, March 22, 2000 4:33 PM
To: Recipients of ABRF List
Subject: 5'-Thiol on oligos

Hi all:

Any ideas of a quick test I can do as qualitative control to "know" that
the thiol can be treated and available for use ?

I use Glen Research's great product 5'-Thiol-Modifier C6 S-S (#
10-1936-xx) added at 5' end of oligos, do DMTO-ON and poly pak cleanup
(no TFA treatment).

I run a 20% 8M urea PAGE for QC prior to giving to customer - but
usually don't do HPLC unless doubts arise.

This works well, but new users sometimes challenge that the oligo is
proper when the science doesn't work well....

What I mean is this...I usually give the client the oligo with DMTO-ON
and the S-S disulfide linkage intact. That way the client can prepare a
portion of the oligo when ready for use, and I like sending it
"protected" and not treated with DTT to reduce the possibility of
oxidative disulfide formation.

I will be running HPLC on some recent samples that "appear to be bad" .
For HPLC I'm going to have 3 samples,
(1) oligo after poly-pak cleanup (therefore DMTO should be ON)
(2) oligo treated with TCA or TFA (therefore DMTO should be OFF)
(3) oligo treated with DTT (therefore thiol will be present)

These should runn differently - right?

But still it won't tell me if the thiol is "useable"....

Any and all suggestions are welcome - even leads to other sources for me
to do the foot work - please

Thanks

Winnell 

-- 
Winnell H. Newman, Director, Nucleic Acids Facility (NAF)
North Carolina State University, Department of Biochemistry
128 Polk Hall, Box 7622
Raleigh, NC 27695-7622

Nucleic Acids Facility Web Page http://www.ncsu.edu/naf
Email = mailto:winnell@ncsu.edu
VOX (919) 515-3463
FAX (919) 515-2047

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