N4C-ethyl-N4C cross-linked DNA: Synthesis and characterization of duplexes with interstrand cross-links of different orientations

Anne M. Noronha, David M. Noll, Christopher J. Wilds, Paul S. Miller

Research output: Contribution to journalArticle

Abstract

The preparation and physical properties of short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N4C-ethyl-N4C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 °C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 °C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 °C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.

Original languageEnglish (US)
Pages (from-to)760-771
Number of pages12
JournalBiochemistry®
Volume41
Issue number3
DOIs
StatePublished - Jan 22 2002

Fingerprint

Oligomers
Base Pairing
Molecular Models
Melting point
DNA
Hydrogen bonds
B-Form DNA
DNA Ligases
Freezing
Propellers
Electrophoresis
Hot Temperature
Thermodynamic stability
Repair
Stabilization
Physical properties
Temperature
Transition Temperature
Hydrogen Bonding
DNA Repair

ASJC Scopus subject areas

  • Biochemistry

Cite this

N4C-ethyl-N4C cross-linked DNA : Synthesis and characterization of duplexes with interstrand cross-links of different orientations. / Noronha, Anne M.; Noll, David M.; Wilds, Christopher J.; Miller, Paul S.

In: Biochemistry®, Vol. 41, No. 3, 22.01.2002, p. 760-771.

Research output: Contribution to journalArticle

Noronha, Anne M. ; Noll, David M. ; Wilds, Christopher J. ; Miller, Paul S. / N4C-ethyl-N4C cross-linked DNA : Synthesis and characterization of duplexes with interstrand cross-links of different orientations. In: Biochemistry®. 2002 ; Vol. 41, No. 3. pp. 760-771.
@article{c53354a033de40f69544ec451ffc4311,
title = "N4C-ethyl-N4C cross-linked DNA: Synthesis and characterization of duplexes with interstrand cross-links of different orientations",
abstract = "The preparation and physical properties of short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give {"}staggered{"} interstrand cross-links were prepared using a novel N4C-ethyl-N4C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 °C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 °C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 °C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.",
author = "Noronha, {Anne M.} and Noll, {David M.} and Wilds, {Christopher J.} and Miller, {Paul S.}",
year = "2002",
month = "1",
day = "22",
doi = "10.1021/bi011610u",
language = "English (US)",
volume = "41",
pages = "760--771",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "3",

}

TY - JOUR

T1 - N4C-ethyl-N4C cross-linked DNA

T2 - Synthesis and characterization of duplexes with interstrand cross-links of different orientations

AU - Noronha, Anne M.

AU - Noll, David M.

AU - Wilds, Christopher J.

AU - Miller, Paul S.

PY - 2002/1/22

Y1 - 2002/1/22

N2 - The preparation and physical properties of short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N4C-ethyl-N4C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 °C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 °C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 °C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.

AB - The preparation and physical properties of short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N4C-ethyl-N4C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 °C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 °C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 °C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.

UR - http://www.scopus.com/inward/record.url?scp=0037154085&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037154085&partnerID=8YFLogxK

U2 - 10.1021/bi011610u

DO - 10.1021/bi011610u

M3 - Article

C2 - 11790097

AN - SCOPUS:0037154085

VL - 41

SP - 760

EP - 771

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 3

ER -