Journal of the American Chemical Society
bly, RhCy3 fluorescence titrations show a significant increase
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ACKNOWLEDGMENT
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in fluorescence intensity in the presence of gDNA isolated
from MMR-deficient cells (HCT116O, DU145, and
SKOV3) compared to gDNA isolated from the MMR-
proficient HCT116N cell line (Figure 5).17,18 Thus, we find
the conjugate to be capable of differentiating the MMR-
deficient versus MMR proficient phenotype, and the results
obtained for RhCy3 correlate well with the reported muta-
tion rates for these cell lines.15,17,19 This differential fluores-
cence enhancement reflects the remarkably high mismatch
specificity of rhodium metalloinsertors; in fact, luminescent
Ru(II) light switch complexes7 do not exhibit comparable
mismatch discrimination in genomic DNA samples.
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We are grateful to the NIH for funding this work. We also
thank the Beckman Institute Laser Resource Center facilities
and Dr. Jay R. Winkler for assistance.
REFERENCES
(1) (a) Loeb, L. A. Cancer Res. 2001, 61, 3230-3239. (b) Bhattacharya, N.
P.; Skandalis, A.; Ganesh, A.; Groden, J.; Meuth, M. Proc. Natl. Acad. Sci.
USA, 1994, 91, 6319-6323.; (c) Strauss, B. S. Mutat. Res. 1999, 437, 195-
203.
(2) Zeglis, B. M.; Pierre, V. C.; Barton, J. K. Chem. Commun. (Camb).
2007, 44, 4565-4579.
(3) Komor, A. C.; Barton, J. K. J. Am. Chem. Soc. 2014, 136, 14160-14172.
(4) Jackson, B. A.; Alekseyev, V. Y.; Barton, J. K. Biochemistry, 1999, 38,
4655-4662.
(5) Zeglis, B. M.; Pierre, V. C.; Kaiser, J. T.; Barton, J. K. Biochemistry,
2009, 48, 4247-4253.
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Natl. Acad. Sci. U. S. A. 2006, 103, 15359-15363.
(7) (a) Boynton, A. N.; Marcélis, L.; Barton, J. K. J. Am. Chem. Soc. 2016
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138, 5020-5023. (b) Boynton, A. N.; Marcélis, L.; McConnell, A. J.; Barton,
J. K. Inorg. Chem. 2017, 56, 8381-8389.
(8) Fung, S. K.; Zou, T.; Cao, B.; Chen, T.; To, W.-P.; Yang, C.; Lok, C.-
N.; Che, C.-M. Nat. Commun. 2016, 7, 1-9.
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Chem. Commun. 2011, 47, 5885-5887. (b) Arambula, J. F.; Ramisetty, S.
HCT116N gDNA
SKOV3 gDNA
HCT116O gDNA
DU145 gDNA
1
0
R.; Baranger, A. M.; Zimmerman, S. C. Proc. Natl. Acad. Sci. USA, 2009
106, 16068-16073.
,
(10) Cyanine’s emissive properties strongly depend on the photo-induced
cis-trans isomerization of the polymethine chain, which can be inhibited
when cyanines are located in a rigid environment. See ref.: Levitus. M.;
Ranjit, S. Q. Rev. of Biophys. 2011, 44, 123-151.
(11) Komor, A. C.; Schneider, C. J.; Weidman, A. G.; Barton, J. K. J. Am.
Chem. Soc. 2012, 123, 19223-19233.
(12) Messina, M. S; Axtell, J. C.; Wang, Y.; Chong, P.; Wixtrom, A. I.;
Kirlikovali, K. O.; Upton, B. M.; Hunter, B. M.; Shafaat, O. S.; Khan, S. I.;
Winkler, J. R.; Gray, H. B.; Alexandrova, A. N.; Maynard, H. D.; Spokoyny,
A. M. J. Am Chem. Soc. 2016, 138, 6952-6955.
(13) Static quenching of the cyanine by the Rh complex is evident for the
conjugate free in solution.
(14) Harvey, B. J.; Levitus, M. J. Fluoresc. 2009, 19, 443-448.
(15) Junicke, H.; Hart, J. R.; Kisko, J.; Glebov, O.; Kirsch, I. R.; Barton, J.
K. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 3737-3742.
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(17) The fluorescence titrations with gDNA in conjunction with the data
obtained from the fluorescence titrations with 27-mer dsDNA provide a
lower estimate of the number of mismatches for MMR-deficient vs. MMR-
proficient cells. Since the 27-mer DNA fluorescence titrations were per-
formed with dsDNA containing only CC mismatches, we assume compa-
rable or lower binding affinities to other (more thermodynamically stable)
mismatches. Based on the fluorescence titrations with genomic samples, we
therefore estimate about 500-fold increase in mismatches in cells that are
MMR-deficient compared to the MMR-proficient analogue (SI, Section 8).
(18) We also note that related metalloinsertors have been shown to bind
abasic sites and single base bulges, which may be relevant to insertions and
deletions that are similarly repaired by MMR machinery. See ref.: Zeglis, B.
M.; Boland, J. A.; Barton, J. K. Biochemistry, 2009, 48, 839-849.
(19) Glaab, W. E.; Tindall, K. R. Carcinogenesis, 1997, 18, 1-8.
0
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600
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1000
[gDNA] (µM)
Figure 5. RhCy3 fluorescence titrations with increasing amounts
of gDNA extracted from HCT116N, HCT116O, SKOV3 and
DU145 cell lines. The fluorescence was measured in Tris buffer
at 25 °C. lEx = 520 nm. [RhCy3] = 1µM. Error bars were calcu-
lated over two replicates. [gDNA] is per base pairs.
Overall these results demonstrate the design and applica-
tion of a Rh metalloinsertor-cyanine conjugate in targeting
mismatched DNA sites with a luminescent reporter of specif-
ic binding. A remarkably high sensitivity of the metalloinser-
tor conjugate to the MMR-deficient phenotype is observed
which points to the application of the conjugate as a new tool
in the early diagnosis of mismatch-repair deficient cancers.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental methods and supporting Figures (S1-S16) (PDF)
AUTHOR INFORMATION
Corresponding Author
*jkbarton@caltech.edu
Funding Sources
No competing financial interests have been declared.
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