Paul, Ananya

Wilson, W. David

English

Georgia State University

Georgia State University
ScholarWorks @ Georgia State University
Chemistry Faculty Publications Department of Chemistry
2-2014
Kinetics and Structures on the Molecular Path to
the Quadruplex Form of the Human Telomere
W. David Wilson
Georgia State University, wdw@gsu.edu
Ananya Paul
Georgia State University, apaul@gsu.edu
Follow this and additional works at: http://scholarworks.gsu.edu/chemistry_facpub
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Recommended Citation
Kinetics and structures on the molecular path to the quadruplex form of the human telomere. W. David Wilson and A. Paul. doi:
http://dx.doi.org/10.1016/j.jmb.2014.02.001
Kinetics and Structures on the Molecular
Path to the Quadruplex Form of the
Human Telomere
W. David Wilson and Ananya Paul
Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
Correspondence to W. David Wilson: Department of Chemistry, Natural Science Center, Georgia State University, 50
Decatur Street, Atlanta, GA 30303, USA.
http://dx.doi.org/10.1016/j.jmb.2014.02.001
Edited by S. A. Woodson
Telomeres, which form the ends of chromosomes
in eukaryotic organisms, have a number of important
functions from protection of the chromosome ends
from degradation to processes in the expression of
some genes, as well as roles in protection of cells
from aging due to loss of DNA in replication and in
cancer development [1–4]. The human telomere
starts at the ends of chromosomal DNA with the
repeat duplex sequence 5 ′ - (TTAGGG)-3 ′
5′-(CCCTAA)-3′ and moves to a single-stranded,
terminal 3′ overhang of the G-rich strand. It has been
known for quite some time that G nucleotides and
G-rich sequences can associate into planar tetrads
that are stabilized by tetrad stacking, multiple
H-bonds and cation interactions (Fig. 1a) [5,6]. The
tetrad structure creates a central hole that is lined by
four oxygen atoms to form an excellent macrocyclic
receptor for K+ ions. Sodium ions and other cations
can also fill the hole, but K+ has the optimum
properties and generally provides the highest stabil-
ity [7–9]. There are a number of different conforma-
tional possibilities for telomere sequences, and two
important examples, found experimentally, are
shown in Fig. 1. Some of the more important features
of the folded states include strand directionality,
parallel or antiparallel, whether the bases are syn or
anti, and the connecting TTA loop arrangements [8–
12].
Wang and Patel determined a structure of a single
G-rich strand of the human telomere, 5 ′-d
[AGGG(TTAGGG)3], (Htelo) in Na
+ solution by
using NMR and molecular dynamics methods
(Fig. 1b) [11]. The structure has three stacked
G-tetrads, as expected from the GGG repeats,
which are formed through folding of the single
strand. The strands in the Na+ structure are
connected into an antiparallel folding pattern by
folding with two lateral loops and one central TTA
loop. This structure creates four grooves, one wide,
two medium and one narrow. The G nucleotides
have both syn and anti orientations. Neidle and
coworkers.[12] solved an X-ray crystal structure of
the Htelo sequence by crystallization from K+ media
in polyethylene glycol and found a very different
structure (Fig. 1c) [12]. In the K+ crystal structure, all
four DNA strands are parallel with the three linking
TTA-connecting sequences oriented in chain-rever-
sal loops along the exterior of the tetrad stack. This
gives the structure a propeller-type appearance
when viewed from the top. As in the Na+ structure,
the G-tetrads are stacked and stabilized by π–π
interactions, H-bonds and cation complexes. All G
nucleotides in this structure are in the anti confor-
mation, and the stacking pattern results in four
almost equivalent grooves. These two very different
structures for Htelo suggested environment-depen-
dent polymorphism of the structures formed from
this sequence [11–15].
Detailed analysis of the solution structure of Htelo in
K+ solution revealed a mixture of structures and
confirmed the polymorphic possibilities of the Htelo
sequence [9,14–18]. Through modification of the
terminal nucleotides, it has been possible to isolate
different single species from the polymorphic ensem-
ble in solution and to determine their structural details
by NMR [16–18]. Interestingly, hybrid structures that
have both parallel and antiparallel strands are a
common observation [19,20]. Two related structures
of particular interest have been referred to asHybrid-1
and Hybrid-2, and their folding patterns are shown in
Fig. 2. As can be seen, both of these have three
parallel strands and one antiparallel strand that are
connected by two lateral loops and one chain-reversal
loop. The two structures are stabilized by different
terminal stacking nucleotides and have the strand
orientations and loops in different positions.
0022-2836/© 2014 The Author. Published by Elsevier Ltd. This is an open access article
under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).
J. Mol. Biol. (2014) 426, 1625–1628
Commentary
In addition to the structures of Htelo, a functionally
very important and interesting question is how does
the G-rich Htelo sequence fold from the open
stranded into the quadruplex conformation [19]?
Protein folding has been a topic of intense interest
and research for over 50 years, and more recently,
the folding mechanisms of complex RNA 3′ struc-
tures have attracted interest. DNA duplex formation
has been less extensively studied, but with the
increasing evidence that complex DNA structures,
such as the quadruplex, exist in cells and have
important functions in both normal and diseased
cells [21], the question of how these structures fold,
as well as the kinetics of folding, has taken on
greater significance. The Sugiyama laboratory [20]
performed extensive molecular dynamic simulations
to model possible quadruplex folding pathways and
were the first to propose a triple-helix intermediate,
but those models were not experimentally tested
until now.
In the Journal of Molecular Biology [19], the authors
have used three different, powerful spectroscopic
probes, circular dichroism, FRET (fluorescence
resonance energy transfer) and 2AP (2-arninopur-
ine) fluorescence changes, to monitor Htelo folding
in the most detailed study to date [19]. The
advantage of using these three is that eachmonitors
a structurally different aspect of Htelo folding. CD
primarily monitors global quadruplex folding since it
is most sensitive to stacking of the G-tetrads.
Different quadruplexes, as well as DNA duplexes
and triplexes, usually have different CD spectra that
provide patterns for their recognition in solution [22].
The FRET signals arise from interaction of probes at
the 5′ and 3′ ends of the G-rich sequence and
monitor the distance between the two ends [23].
Fluorescence from 2AP arises from replacement of
an A base in the quadruplex TTA loops, and
fluorescence from this group can provide a monitor
of folding of each loop [24]. With these probes, the
authors were able to monitor the kinetics of folding at
both the global level and at specific sites. They used
three DNA sequences, the d[AGGG(TTAGGG)3]
natural sequence (Tel22), and the two hybrid
Fig. 1. Structures for (a) a G-quadruplex tetrad interaction, (b) a basket-type intramolecular G-quadruplex found in Na+
solution [11] and (c) a propeller-type parallel-stranded intramolecular G-quadruplex found in the presence of K+ in the
crystalline state [12].
Fig. 2. Schematic outline of folding topologies of the Hybrid-1 (a) and Hybrid-2 (b) intramolecular human telomere
G-quadruplexes in K+ solution.
1626 Kinetics and Structures on the Molecular Path
sequences found by NMR analysis to give primarily
single structures (Fig. 2), to follow Htelo folding.
Interestingly, in spite of their simpler, ensemble of
structures, Hybrid-1 and Hybrid-2 follow a similar
complex folding pathway to the natural sequence.
Folding of all these sequences, which can be
induced by adding K+ to U, appears to follow a
multistep mechanism that requires at least three
intermediates (Scheme 1): where U represents
unfolded oligonucleotide conformers; I1, I2 and I3
are folding intermediates and F is the final folded
state or ensemble of states. By using the spectro-
scopic methods described above along with SVD
(single value decomposition) analysis, the authors
have been able not only to define the number of
species but also to propose structures for each
intermediate that fit well into what we know about
DNA duplex and quadruplex folding in general.
The first intermediate, I1, forms very rapidly and is
proposed to be dominated by hairpin conformations
formed by folding of the G-rich strand to allow G–G
interactions that are relatively weak and dynamic
(Fig. 3). The I2 folded state forms next, in under 10 s,
but the authors were able to obtain a CD spectrum
for I2 by conducting short-time (2 s) CD kinetics
studies as a function of wavelength. This intermedi-
ate spectrum is very informative and is characteristic
of an antiparallel structure, such as the Na+ structure
in Fig. 1, but the precise conformation cannot be
defined by CD. It seems likely that the state is an
ensemble of antiparallel structures.
The steps in conversion of I2 → I3 → F are
significantly slower and, as a result, can be
evaluated by manual mixing of U with K+. CD
spectra were collected after the initial 10-s folding up
to 104 s. SVD and fitting analyses in this time region
gave a time constant of ~3700 s for I2 → I3 and a
faster final folding of I3 → F with a time constant of
~160 s. From SVD analysis, the significant spectral
species could be associated with the most likely
structure for each intermediate. As noted above, I2 is
likely an antiparallel G4 structure while the SVD
results suggest that I3 is most likely a triple-helix
conformation that folds into the final hybrid confor-
mation in K+ (Figs. 3 and 12 in Ref. [19]).
Kinetic results monitored by FRET and by replac-
ing the 5′ A and the A in each of the three loops with
2AP support the complex kinetic Scheme 1 pro-
posed from CD experiments as well as the proposed
structures. All of these methods share relaxation
times of ~0.2, 21 and 1300 s. The unfolded state
gives an ensemble of dynamic hairpin duplexes that
can fold in a direct manner into antiparallel quad-
ruplex structures (such as the “chair” conformation in
Fig. 3). Although the antiparallel conformation
cannot be exactly specified, the chair conformation
seems most likely since such structures can be
formed by simple folding of initial hairpins. Opening
of the chair from either the 5′ or the 3′ end can give
the proposed triple-helical structures. The triple
helices allow refolding into the alternative orienta-
tions of either Hybrid-1 or Hybrid-2 [19]. Opening of
the relatively stable antiparallel structure (I2) to give
the triple helix has high activation energy and
represents a significant kinetic barrier to formation
of the final folded hybrid quadruplex. The anti–syn
conversions of some nucleotides required for the
final structures are also analogous to the cis–trans
proline conversions required in folding of some
proteins. The unfolding of the hybrid quadruplex
structures was followed by trapping the unfolded
state with the complementary C-rich strand since
formation of the trapped duplex is faster than
unfolding of the quadruplex F state. Complex,
multiphasic kinetics was also observed in unfolding
and generally supports the proposed mechanism in
Scheme 1 and Fig. 3.
To test their proposed conformational scheme, the
authors conducted constrained simulation studies of
the folding process and they present movies of the
folding process in Supplementary Materials [19].
Scheme 1. Folding pathway to the final structure (F) of
the human telomere quadruplex in solution.
Fig. 3. Proposed folding mechanisms for the formation of the human telomere G-quadruplex.
1627Kinetics and Structures on the Molecular Path
These simulations provide strong support for the
proposed conformational conversions and give very
useful visual assistance in understanding the possi-
ble folding steps in the kinetic pathway to the final
quadruplex. It will be of great interest to conduct
similar detailed kinetics studies on DNA quadru-
plexes of different structure from Htelo.
References
[1] Blackburn EH. Structure and function of telomers. Nature
1991;350:569–73.
[2] Rhodes D, Giraldo R. Telomere structure and function. Curr
Opin Struct Biol 1995;5:311–22.
[3] O'Sullivan RJ, Karlseder J. Telomeres: protecting chromo-
somes against genome instability. Nat Rev Mol Cell Biol
2010;11:171–81.
[4] Cech TR. Life at the end of chromosome: telomers and
telomerase. Angew Chem Int Ed 2000;39:34–43.
[5] Blackburn EH, Szostak JW. The molecular structure of
cent romers and te lomers. Annu Rev Biochem
1984;53:163–94.
[6] Meyne J, Ratliff RL, Moyzis RK. Conservation of the human
telomere sequence (TTAGGG)n among vertebrates. Proc
Natl Acad Sci U S A 1989;86:7049–53.
[7] Hardin CC, Henderson E, Watson T, Prosser JK. Monova-
lent cation induced structural transitions in telomeric DNAs:
G-DNA folding intermediates. Biochemistry 1991;30:
4460–72.
[8] Gray RD, Chaires JB. Kinetics and mechanism of K+- and
Na+-induced folding of models of human telomeric DNA into
G-quadruplex structures. Nucleic Acids Res 2008;36:
4191–203.
[9] Jixun D, Megan C, Chandanamali P, Roger AJ, Danzhou Y.
Structure of the hybrid-2 type intramolecular human telomeric
G-quadruplex in K+ solution: insights into structure polymor-
phism of the human telomeric sequence. Nucleic Acids Res
2007;35:4927–40.
[10] Yang D, Okamoto K. Structural insights into G-quadruplexes:
towards new anticancer drugs. Future Med Chem
2010;2:619–46.
[11] Wang Y, Patel DJ. Solution structure of the human telomeric
repeat D[Ag3(T2Ag3)3] G-tetraplex. Structure 1993;1:263–82.
[12] Parkinson GN, Lee MP, Neidle S. Crystal structure of parallel
quadruplexes from human telomeric DNA. Nature
2002;417:876–80.
[13] Li J, Correia JJ, Wang L, Trent JO, Chaires JB. Not so crystal
clear: the structure of the human telomere G-quadruplex in
solution differs from that present in a crystal. Nucleic Acids
Res 2005;33:4649–59.
[14] Dai JX, Carver M, YangDZ. Polymorphism of human telomeric
quadruplex structures. Biochimie 2008;90:1172–83.
[15] Buscaglia R, Miller MC, Dean WL, Gray RD, Lane AN, Trent
JO, et al. Polyethylene glycol binding alters human telomere
G-quadruplex structure by conformational selection. Nucleic
Acids Res 2013;41:7934–46.
[16] Phan AT, Luu KN, Patel DJ. Different loop arrangements of
intramolecular human telomeric (3 + 1) G-quadruplexes in
K+ solution. Nucleic Acids Res 2006;34:5715–9.
[17] Zhang Z, Dai J, Veliath E, Jones RA, Yang D. Structure of a
two-G-tetrad intramolecular G-quadruplex formed by a
variant human telomeric sequence in K+ solution: insights
into the interconversion of human telomeric G-quadruplex
structures. Nucleic Acids Res 2010;38:1009–21.
[18] Ambrus A, Chen D, Dai J, Bialis T, Jones RA, Yang D. Human
telomeric sequence forms a hybrid-type intramolecular G-
quadruplex structure with mixed parallel/antiparallel strands in
potassium solution. Nucleic Acids Res 2006;34:2723–35.
[19] Gray RD, Trent JO, Chaires JB. Folding and unfolding
pathways of the human telomeric G-quadruplex. J Mol Biol
2014;426. http://dx.doi.org/10.1016/j.jmb.2014.01.009.
[20] Mashimo T, Yagi H, Sannohe Y, Rajendran A, Sugiyama H.
Folding pathways of human telomeric type-1 and type-2 G-
quadruplex structures. J Am Chem Soc 2010;132:14910–8.
[21] Biffi G, Tannahill D, McCafferty J, Balasubramanian S.
Quantitative visualization of DNA G-quadruplex structures
in human cells. Nat Chem 2013;5:182–6.
[22] Karsisiotis AI, Hessari NM, Novellino E, Spada GP,
Randazzo A, Webba da Silva M. Topological characteriza-
tion of nucleic acid G-quadruplexes by UV absorption and
circular dichroism. Angew Chem Int Ed 2011;50:10645–8.
[23] TorimuraM, Kurata S, Yamada K, Yokomaku T, Kamagata Y,
Kanagawa T, et al. Fluorescence-quenching phenomenon by
photoinduced electron transfer between a fluorescent dye
and a nucleotide base. Anal Sci 2001;17:155–60.
[24] Gray RD, Petraccone L, Buscaglia R, Chaires JB. 2-
Aminopurine as a probe for quadruplex loop structures.
Methods Mol Biol 2010;608:121–36.
1628 Kinetics and Structures on the Molecular Path


Kinetics and Structures on the Molecular Path to the Quadruplex Form of the Human Telomere

David Wilson, W.

Elsevier - Publisher Connector 

CommentaryW. David Wilso0022-2836/© 2014 The Au
under the CC BY-NC-SA lKinetics and Structures on the Molecular
Path to the Quadruplex Form of the
Human Telomeren and Ananya PaulDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
Correspondence to W. David Wilson: Department of Chemistry, Natural Science Center, Georgia State University, 50
Decatur Street, Atlanta, GA 30303, USA.
http://dx.doi.org/10.1016/j.jmb.2014.02.001
Edited by S. A. WoodsonTelomeres, which form the ends of chromosomes
in eukaryotic organisms, have a number of important
functions from protection of the chromosome ends
from degradation to processes in the expression of
some genes, as well as roles in protection of cells
from aging due to loss of DNA in replication and in
cancer development [1–4]. The human telomere
starts at the ends of chromosomal DNA with the
repeat duplex sequence 5 ′ - (TTAGGG)-3 ′
5′-(CCCTAA)-3′ and moves to a single-stranded,
terminal 3′ overhang of the G-rich strand. It has been
known for quite some time that G nucleotides and
G-rich sequences can associate into planar tetrads
that are stabilized by tetrad stacking, multiple
H-bonds and cation interactions (Fig. 1a) [5,6]. The
tetrad structure creates a central hole that is lined by
four oxygen atoms to form an excellent macrocyclic
receptor for K+ ions. Sodium ions and other cations
can also fill the hole, but K+ has the optimum
properties and generally provides the highest stabil-
ity [7–9]. There are a number of different conforma-
tional possibilities for telomere sequences, and two
important examples, found experimentally, are
shown in Fig. 1. Some of the more important features
of the folded states include strand directionality,
parallel or antiparallel, whether the bases are syn or
anti, and the connecting TTA loop arrangements [8–
12].
Wang and Patel determined a structure of a single
G-rich strand of the human telomere, 5 ′-d
[AGGG(TTAGGG)3], (Htelo) in Na
+ solution by
using NMR and molecular dynamics methods
(Fig. 1b) [11]. The structure has three stacked
G-tetrads, as expected from the GGG repeats,
which are formed through folding of the single
strand. The strands in the Na+ structure are
connected into an antiparallel folding pattern by
folding with two lateral loops and one central TTAthor. Published by Elsevier Ltd. This is an o
icense (http://creativecommons.org/licenses/bloop. This structure creates four grooves, one wide,
two medium and one narrow. The G nucleotides
have both syn and anti orientations. Neidle and
coworkers.[12] solved an X-ray crystal structure of
the Htelo sequence by crystallization from K+ media
in polyethylene glycol and found a very different
structure (Fig. 1c) [12]. In the K+ crystal structure, all
four DNA strands are parallel with the three linking
TTA-connecting sequences oriented in chain-rever-
sal loops along the exterior of the tetrad stack. This
gives the structure a propeller-type appearance
when viewed from the top. As in the Na+ structure,
the G-tetrads are stacked and stabilized by π–π
interactions, H-bonds and cation complexes. All G
nucleotides in this structure are in the anti confor-
mation, and the stacking pattern results in four
almost equivalent grooves. These two very different
structures for Htelo suggested environment-depen-
dent polymorphism of the structures formed from
this sequence [11–15].
Detailed analysis of the solution structure of Htelo in
K+ solution revealed a mixture of structures and
confirmed the polymorphic possibilities of the Htelo
sequence [9,14–18]. Through modification of the
terminal nucleotides, it has been possible to isolate
different single species from the polymorphic ensem-
ble in solution and to determine their structural details
by NMR [16–18]. Interestingly, hybrid structures that
have both parallel and antiparallel strands are a
common observation [19,20]. Two related structures
of particular interest have been referred to asHybrid-1
and Hybrid-2, and their folding patterns are shown in
Fig. 2. As can be seen, both of these have three
parallel strands and one antiparallel strand that are
connected by two lateral loops and one chain-reversal
loop. The two structures are stabilized by different
terminal stacking nucleotides and have the strand
orientations and loops in different positions.pen access article
y-nc-sa/3.0/).
J. Mol. Biol. (2014) 426, 1625–1628
Fig. 1. Structures for (a) a G-quadruplex tetrad interaction, (b) a basket-type intramolecular G-quadruplex found in Na+
solution [11] and (c) a propeller-type parallel-stranded intramolecular G-quadruplex found in the presence of K+ in the
crystalline state [12].
1626 Kinetics and Structures on the Molecular PathIn addition to the structures of Htelo, a functionally
very important and interesting question is how does
the G-rich Htelo sequence fold from the open
stranded into the quadruplex conformation [19]?
Protein folding has been a topic of intense interest
and research for over 50 years, and more recently,
the folding mechanisms of complex RNA 3′ struc-
tures have attracted interest. DNA duplex formation
has been less extensively studied, but with the
increasing evidence that complex DNA structures,
such as the quadruplex, exist in cells and have
important functions in both normal and diseased
cells [21], the question of how these structures fold,
as well as the kinetics of folding, has taken on
greater significance. The Sugiyama laboratory [20]
performed extensive molecular dynamic simulations
to model possible quadruplex folding pathways and
were the first to propose a triple-helix intermediate,
but those models were not experimentally tested
until now.
In the Journal of Molecular Biology [19], the authors
have used three different, powerful spectroscopicFig. 2. Schematic outline of folding topologies of the Hybr
G-quadruplexes in K+ solution.probes, circular dichroism, FRET (fluorescence
resonance energy transfer) and 2AP (2-arninopur-
ine) fluorescence changes, to monitor Htelo folding
in the most detailed study to date [19]. The
advantage of using these three is that eachmonitors
a structurally different aspect of Htelo folding. CD
primarily monitors global quadruplex folding since it
is most sensitive to stacking of the G-tetrads.
Different quadruplexes, as well as DNA duplexes
and triplexes, usually have different CD spectra that
provide patterns for their recognition in solution [22].
The FRET signals arise from interaction of probes at
the 5′ and 3′ ends of the G-rich sequence and
monitor the distance between the two ends [23].
Fluorescence from 2AP arises from replacement of
an A base in the quadruplex TTA loops, and
fluorescence from this group can provide a monitor
of folding of each loop [24]. With these probes, the
authors were able to monitor the kinetics of folding at
both the global level and at specific sites. They used
three DNA sequences, the d[AGGG(TTAGGG)3]
natural sequence (Tel22), and the two hybridid-1 (a) and Hybrid-2 (b) intramolecular human telomere
Scheme 1. Folding pathway to the final structure (F) of
the human telomere quadruplex in solution.
1627Kinetics and Structures on the Molecular Pathsequences found by NMR analysis to give primarily
single structures (Fig. 2), to follow Htelo folding.
Interestingly, in spite of their simpler, ensemble of
structures, Hybrid-1 and Hybrid-2 follow a similar
complex folding pathway to the natural sequence.
Folding of all these sequences, which can be
induced by adding K+ to U, appears to follow a
multistep mechanism that requires at least three
intermediates (Scheme 1): where U represents
unfolded oligonucleotide conformers; I1, I2 and I3
are folding intermediates and F is the final folded
state or ensemble of states. By using the spectro-
scopic methods described above along with SVD
(single value decomposition) analysis, the authors
have been able not only to define the number of
species but also to propose structures for each
intermediate that fit well into what we know about
DNA duplex and quadruplex folding in general.
The first intermediate, I1, forms very rapidly and is
proposed to be dominated by hairpin conformations
formed by folding of the G-rich strand to allow G–G
interactions that are relatively weak and dynamic
(Fig. 3). The I2 folded state forms next, in under 10 s,
but the authors were able to obtain a CD spectrum
for I2 by conducting short-time (2 s) CD kinetics
studies as a function of wavelength. This intermedi-
ate spectrum is very informative and is characteristic
of an antiparallel structure, such as the Na+ structure
in Fig. 1, but the precise conformation cannot be
defined by CD. It seems likely that the state is an
ensemble of antiparallel structures.
The steps in conversion of I2 → I3 → F are
significantly slower and, as a result, can be
evaluated by manual mixing of U with K+. CD
spectra were collected after the initial 10-s folding up
to 104 s. SVD and fitting analyses in this time regionFig. 3. Proposed folding mechanisms for the fogave a time constant of ~3700 s for I2 → I3 and a
faster final folding of I3 → F with a time constant of
~160 s. From SVD analysis, the significant spectral
species could be associated with the most likely
structure for each intermediate. As noted above, I2 is
likely an antiparallel G4 structure while the SVD
results suggest that I3 is most likely a triple-helix
conformation that folds into the final hybrid confor-
mation in K+ (Figs. 3 and 12 in Ref. [19]).
Kinetic results monitored by FRET and by replac-
ing the 5′ A and the A in each of the three loops with
2AP support the complex kinetic Scheme 1 pro-
posed from CD experiments as well as the proposed
structures. All of these methods share relaxation
times of ~0.2, 21 and 1300 s. The unfolded state
gives an ensemble of dynamic hairpin duplexes that
can fold in a direct manner into antiparallel quad-
ruplex structures (such as the “chair” conformation in
Fig. 3). Although the antiparallel conformation
cannot be exactly specified, the chair conformation
seems most likely since such structures can be
formed by simple folding of initial hairpins. Opening
of the chair from either the 5′ or the 3′ end can give
the proposed triple-helical structures. The triple
helices allow refolding into the alternative orienta-
tions of either Hybrid-1 or Hybrid-2 [19]. Opening of
the relatively stable antiparallel structure (I2) to give
the triple helix has high activation energy and
represents a significant kinetic barrier to formation
of the final folded hybrid quadruplex. The anti–syn
conversions of some nucleotides required for the
final structures are also analogous to the cis–trans
proline conversions required in folding of some
proteins. The unfolding of the hybrid quadruplex
structures was followed by trapping the unfolded
state with the complementary C-rich strand since
formation of the trapped duplex is faster than
unfolding of the quadruplex F state. Complex,
multiphasic kinetics was also observed in unfolding
and generally supports the proposed mechanism in
Scheme 1 and Fig. 3.
To test their proposed conformational scheme, the
authors conducted constrained simulation studies of
the folding process and they present movies of the
folding process in Supplementary Materials [19].rmation of the human telomere G-quadruplex.
1628 Kinetics and Structures on the Molecular PathThese simulations provide strong support for the
proposed conformational conversions and give very
useful visual assistance in understanding the possi-
ble folding steps in the kinetic pathway to the final
quadruplex. It will be of great interest to conduct
similar detailed kinetics studies on DNA quadru-
plexes of different structure from Htelo.References
[1] Blackburn EH. Structure and function of telomers. Nature
1991;350:569–73.
[2] Rhodes D, Giraldo R. Telomere structure and function. Curr
Opin Struct Biol 1995;5:311–22.
[3] O'Sullivan RJ, Karlseder J. Telomeres: protecting chromo-
somes against genome instability. Nat Rev Mol Cell Biol
2010;11:171–81.
[4] Cech TR. Life at the end of chromosome: telomers and
telomerase. Angew Chem Int Ed 2000;39:34–43.
[5] Blackburn EH, Szostak JW. The molecular structure of
cent romers and te lomers. Annu Rev Biochem
1984;53:163–94.
[6] Meyne J, Ratliff RL, Moyzis RK. Conservation of the human
telomere sequence (TTAGGG)n among vertebrates. Proc
Natl Acad Sci U S A 1989;86:7049–53.
[7] Hardin CC, Henderson E, Watson T, Prosser JK. Monova-
lent cation induced structural transitions in telomeric DNAs:
G-DNA folding intermediates. Biochemistry 1991;30:
4460–72.
[8] Gray RD, Chaires JB. Kinetics and mechanism of K+- and
Na+-induced folding of models of human telomeric DNA into
G-quadruplex structures. Nucleic Acids Res 2008;36:
4191–203.
[9] Jixun D, Megan C, Chandanamali P, Roger AJ, Danzhou Y.
Structure of the hybrid-2 type intramolecular human telomeric
G-quadruplex in K+ solution: insights into structure polymor-
phism of the human telomeric sequence. Nucleic Acids Res
2007;35:4927–40.
[10] Yang D, Okamoto K. Structural insights into G-quadruplexes:
towards new anticancer drugs. Future Med Chem
2010;2:619–46.
[11] Wang Y, Patel DJ. Solution structure of the human telomeric
repeat D[Ag3(T2Ag3)3] G-tetraplex. Structure 1993;1:263–82.[12] Parkinson GN, Lee MP, Neidle S. Crystal structure of parallel
quadruplexes from human telomeric DNA. Nature
2002;417:876–80.
[13] Li J, Correia JJ, Wang L, Trent JO, Chaires JB. Not so crystal
clear: the structure of the human telomere G-quadruplex in
solution differs from that present in a crystal. Nucleic Acids
Res 2005;33:4649–59.
[14] Dai JX, Carver M, YangDZ. Polymorphism of human telomeric
quadruplex structures. Biochimie 2008;90:1172–83.
[15] Buscaglia R, Miller MC, Dean WL, Gray RD, Lane AN, Trent
JO, et al. Polyethylene glycol binding alters human telomere
G-quadruplex structure by conformational selection. Nucleic
Acids Res 2013;41:7934–46.
[16] Phan AT, Luu KN, Patel DJ. Different loop arrangements of
intramolecular human telomeric (3 + 1) G-quadruplexes in
K+ solution. Nucleic Acids Res 2006;34:5715–9.
[17] Zhang Z, Dai J, Veliath E, Jones RA, Yang D. Structure of a
two-G-tetrad intramolecular G-quadruplex formed by a
variant human telomeric sequence in K+ solution: insights
into the interconversion of human telomeric G-quadruplex
structures. Nucleic Acids Res 2010;38:1009–21.
[18] Ambrus A, Chen D, Dai J, Bialis T, Jones RA, Yang D. Human
telomeric sequence forms a hybrid-type intramolecular G-
quadruplex structure with mixed parallel/antiparallel strands in
potassium solution. Nucleic Acids Res 2006;34:2723–35.
[19] Gray RD, Trent JO, Chaires JB. Folding and unfolding
pathways of the human telomeric G-quadruplex. J Mol Biol
2014;426. http://dx.doi.org/10.1016/j.jmb.2014.01.009.
[20] Mashimo T, Yagi H, Sannohe Y, Rajendran A, Sugiyama H.
Folding pathways of human telomeric type-1 and type-2 G-
quadruplex structures. J Am Chem Soc 2010;132:14910–8.
[21] Biffi G, Tannahill D, McCafferty J, Balasubramanian S.
Quantitative visualization of DNA G-quadruplex structures
in human cells. Nat Chem 2013;5:182–6.
[22] Karsisiotis AI, Hessari NM, Novellino E, Spada GP,
Randazzo A, Webba da Silva M. Topological characteriza-
tion of nucleic acid G-quadruplexes by UV absorption and
circular dichroism. Angew Chem Int Ed 2011;50:10645–8.
[23] TorimuraM, Kurata S, Yamada K, Yokomaku T, Kamagata Y,
Kanagawa T, et al. Fluorescence-quenching phenomenon by
photoinduced electron transfer between a fluorescent dye
and a nucleotide base. Anal Sci 2001;17:155–60.
[24] Gray RD, Petraccone L, Buscaglia R, Chaires JB. 2-
Aminopurine as a probe for quadruplex loop structures.
Methods Mol Biol 2010;608:121–36.


Kinetics and Structures on the Molecular Path to the Quadruplex Form of the Human Telomere 

ScholarWorks @ Georgia State University

Georgia State UniversityScholarWorks @ Georgia State UniversityChemistry Faculty Publications Department of Chemistry2-2014Kinetics and Structures on the Molecular Path tothe Quadruplex Form of the Human TelomereW. David WilsonGeorgia State University, wdw@gsu.eduAnanya PaulGeorgia State University, apaul@gsu.eduFollow this and additional works at: http://scholarworks.gsu.edu/chemistry_facpubPart of the Chemistry CommonsThis Article is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has beenaccepted for inclusion in Chemistry Faculty Publications by an authorized administrator of ScholarWorks @ Georgia State University. For moreinformation, please contact scholarworks@gsu.edu.Recommended CitationKinetics and structures on the molecular path to the quadruplex form of the human telomere. W. David Wilson and A. Paul. doi:http://dx.doi.org/10.1016/j.jmb.2014.02.001Kinetics and Structures on the MolecularPath to the Quadruplex Form of theHuman TelomereW. David Wilson and Ananya PaulDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USACorrespondence to W. David Wilson: Department of Chemistry, Natural Science Center, Georgia State University, 50Decatur Street, Atlanta, GA 30303, USA.http://dx.doi.org/10.1016/j.jmb.2014.02.001Edited by S. A. WoodsonTelomeres, which form the ends of chromosomesin eukaryotic organisms, have a number of importantfunctions from protection of the chromosome endsfrom degradation to processes in the expression ofsome genes, as well as roles in protection of cellsfrom aging due to loss of DNA in replication and incancer development [1–4]. The human telomerestarts at the ends of chromosomal DNA with therepeat duplex sequence 5 ′ - (TTAGGG)-3 ′5′-(CCCTAA)-3′ and moves to a single-stranded,terminal 3′ overhang of the G-rich strand. It has beenknown for quite some time that G nucleotides andG-rich sequences can associate into planar tetradsthat are stabilized by tetrad stacking, multipleH-bonds and cation interactions (Fig. 1a) [5,6]. Thetetrad structure creates a central hole that is lined byfour oxygen atoms to form an excellent macrocyclicreceptor for K+ ions. Sodium ions and other cationscan also fill the hole, but K+ has the optimumproperties and generally provides the highest stabil-ity [7–9]. There are a number of different conforma-tional possibilities for telomere sequences, and twoimportant examples, found experimentally, areshown in Fig. 1. Some of the more important featuresof the folded states include strand directionality,parallel or antiparallel, whether the bases are syn oranti, and the connecting TTA loop arrangements [8–12].Wang and Patel determined a structure of a singleG-rich strand of the human telomere, 5 ′-d[AGGG(TTAGGG)3], (Htelo) in Na+ solution byusing NMR and molecular dynamics methods(Fig. 1b) [11]. The structure has three stackedG-tetrads, as expected from the GGG repeats,which are formed through folding of the singlestrand. The strands in the Na+ structure areconnected into an antiparallel folding pattern byfolding with two lateral loops and one central TTAloop. This structure creates four grooves, one wide,two medium and one narrow. The G nucleotideshave both syn and anti orientations. Neidle andcoworkers.[12] solved an X-ray crystal structure ofthe Htelo sequence by crystallization from K+ mediain polyethylene glycol and found a very differentstructure (Fig. 1c) [12]. In the K+ crystal structure, allfour DNA strands are parallel with the three linkingTTA-connecting sequences oriented in chain-rever-sal loops along the exterior of the tetrad stack. Thisgives the structure a propeller-type appearancewhen viewed from the top. As in the Na+ structure,the G-tetrads are stacked and stabilized by π–πinteractions, H-bonds and cation complexes. All Gnucleotides in this structure are in the anti confor-mation, and the stacking pattern results in fouralmost equivalent grooves. These two very differentstructures for Htelo suggested environment-depen-dent polymorphism of the structures formed fromthis sequence [11–15].Detailed analysis of the solution structure of Htelo inK+ solution revealed a mixture of structures andconfirmed the polymorphic possibilities of the Htelosequence [9,14–18]. Through modification of theterminal nucleotides, it has been possible to isolatedifferent single species from the polymorphic ensem-ble in solution and to determine their structural detailsby NMR [16–18]. Interestingly, hybrid structures thathave both parallel and antiparallel strands are acommon observation [19,20]. Two related structuresof particular interest have been referred to asHybrid-1and Hybrid-2, and their folding patterns are shown inFig. 2. As can be seen, both of these have threeparallel strands and one antiparallel strand that areconnected by two lateral loops and one chain-reversalloop. The two structures are stabilized by differentterminal stacking nucleotides and have the strandorientations and loops in different positions.0022-2836/© 2014 The Author. Published by Elsevier Ltd. This is an open access articleunder the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).J. Mol. Biol. (2014) 426, 1625–1628CommentaryIn addition to the structures of Htelo, a functionallyvery important and interesting question is how doesthe G-rich Htelo sequence fold from the openstranded into the quadruplex conformation [19]?Protein folding has been a topic of intense interestand research for over 50 years, and more recently,the folding mechanisms of complex RNA 3′ struc-tures have attracted interest. DNA duplex formationhas been less extensively studied, but with theincreasing evidence that complex DNA structures,such as the quadruplex, exist in cells and haveimportant functions in both normal and diseasedcells [21], the question of how these structures fold,as well as the kinetics of folding, has taken ongreater significance. The Sugiyama laboratory [20]performed extensive molecular dynamic simulationsto model possible quadruplex folding pathways andwere the first to propose a triple-helix intermediate,but those models were not experimentally testeduntil now.In the Journal of Molecular Biology [19], the authorshave used three different, powerful spectroscopicprobes, circular dichroism, FRET (fluorescenceresonance energy transfer) and 2AP (2-arninopur-ine) fluorescence changes, to monitor Htelo foldingin the most detailed study to date [19]. Theadvantage of using these three is that eachmonitorsa structurally different aspect of Htelo folding. CDprimarily monitors global quadruplex folding since itis most sensitive to stacking of the G-tetrads.Different quadruplexes, as well as DNA duplexesand triplexes, usually have different CD spectra thatprovide patterns for their recognition in solution [22].The FRET signals arise from interaction of probes atthe 5′ and 3′ ends of the G-rich sequence andmonitor the distance between the two ends [23].Fluorescence from 2AP arises from replacement ofan A base in the quadruplex TTA loops, andfluorescence from this group can provide a monitorof folding of each loop [24]. With these probes, theauthors were able to monitor the kinetics of folding atboth the global level and at specific sites. They usedthree DNA sequences, the d[AGGG(TTAGGG)3]natural sequence (Tel22), and the two hybridFig. 1. Structures for (a) a G-quadruplex tetrad interaction, (b) a basket-type intramolecular G-quadruplex found in Na+solution [11] and (c) a propeller-type parallel-stranded intramolecular G-quadruplex found in the presence of K+ in thecrystalline state [12].Fig. 2. Schematic outline of folding topologies of the Hybrid-1 (a) and Hybrid-2 (b) intramolecular human telomereG-quadruplexes in K+ solution.1626 Kinetics and Structures on the Molecular Pathsequences found by NMR analysis to give primarilysingle structures (Fig. 2), to follow Htelo folding.Interestingly, in spite of their simpler, ensemble ofstructures, Hybrid-1 and Hybrid-2 follow a similarcomplex folding pathway to the natural sequence.Folding of all these sequences, which can beinduced by adding K+ to U, appears to follow amultistep mechanism that requires at least threeintermediates (Scheme 1): where U representsunfolded oligonucleotide conformers; I1, I2 and I3are folding intermediates and F is the final foldedstate or ensemble of states. By using the spectro-scopic methods described above along with SVD(single value decomposition) analysis, the authorshave been able not only to define the number ofspecies but also to propose structures for eachintermediate that fit well into what we know aboutDNA duplex and quadruplex folding in general.The first intermediate, I1, forms very rapidly and isproposed to be dominated by hairpin conformationsformed by folding of the G-rich strand to allow G–Ginteractions that are relatively weak and dynamic(Fig. 3). The I2 folded state forms next, in under 10 s,but the authors were able to obtain a CD spectrumfor I2 by conducting short-time (2 s) CD kineticsstudies as a function of wavelength. This intermedi-ate spectrum is very informative and is characteristicof an antiparallel structure, such as the Na+ structurein Fig. 1, but the precise conformation cannot bedefined by CD. It seems likely that the state is anensemble of antiparallel structures.The steps in conversion of I2 → I3 → F aresignificantly slower and, as a result, can beevaluated by manual mixing of U with K+. CDspectra were collected after the initial 10-s folding upto 104 s. SVD and fitting analyses in this time regiongave a time constant of ~3700 s for I2 → I3 and afaster final folding of I3 → F with a time constant of~160 s. From SVD analysis, the significant spectralspecies could be associated with the most likelystructure for each intermediate. As noted above, I2 islikely an antiparallel G4 structure while the SVDresults suggest that I3 is most likely a triple-helixconformation that folds into the final hybrid confor-mation in K+ (Figs. 3 and 12 in Ref. [19]).Kinetic results monitored by FRET and by replac-ing the 5′ A and the A in each of the three loops with2AP support the complex kinetic Scheme 1 pro-posed from CD experiments as well as the proposedstructures. All of these methods share relaxationtimes of ~0.2, 21 and 1300 s. The unfolded stategives an ensemble of dynamic hairpin duplexes thatcan fold in a direct manner into antiparallel quad-ruplex structures (such as the “chair” conformation inFig. 3). Although the antiparallel conformationcannot be exactly specified, the chair conformationseems most likely since such structures can beformed by simple folding of initial hairpins. Openingof the chair from either the 5′ or the 3′ end can givethe proposed triple-helical structures. The triplehelices allow refolding into the alternative orienta-tions of either Hybrid-1 or Hybrid-2 [19]. Opening ofthe relatively stable antiparallel structure (I2) to givethe triple helix has high activation energy andrepresents a significant kinetic barrier to formationof the final folded hybrid quadruplex. The anti–synconversions of some nucleotides required for thefinal structures are also analogous to the cis–transproline conversions required in folding of someproteins. The unfolding of the hybrid quadruplexstructures was followed by trapping the unfoldedstate with the complementary C-rich strand sinceformation of the trapped duplex is faster thanunfolding of the quadruplex F state. Complex,multiphasic kinetics was also observed in unfoldingand generally supports the proposed mechanism inScheme 1 and Fig. 3.To test their proposed conformational scheme, theauthors conducted constrained simulation studies ofthe folding process and they present movies of thefolding process in Supplementary Materials [19].Scheme 1. Folding pathway to the final structure (F) ofthe human telomere quadruplex in solution.Fig. 3. Proposed folding mechanisms for the formation of the human telomere G-quadruplex.1627Kinetics and Structures on the Molecular PathThese simulations provide strong support for theproposed conformational conversions and give veryuseful visual assistance in understanding the possi-ble folding steps in the kinetic pathway to the finalquadruplex. It will be of great interest to conductsimilar detailed kinetics studies on DNA quadru-plexes of different structure from Htelo.References[1] Blackburn EH. Structure and function of telomers. Nature1991;350:569–73.[2] Rhodes D, Giraldo R. Telomere structure and function. CurrOpin Struct Biol 1995;5:311–22.[3] O'Sullivan RJ, Karlseder J. Telomeres: protecting chromo-somes against genome instability. Nat Rev Mol Cell Biol2010;11:171–81.[4] Cech TR. Life at the end of chromosome: telomers andtelomerase. Angew Chem Int Ed 2000;39:34–43.[5] Blackburn EH, Szostak JW. The molecular structure ofcent romers and te lomers. Annu Rev Biochem1984;53:163–94.[6] Meyne J, Ratliff RL, Moyzis RK. Conservation of the humantelomere sequence (TTAGGG)n among vertebrates. ProcNatl Acad Sci U S A 1989;86:7049–53.[7] Hardin CC, Henderson E, Watson T, Prosser JK. Monova-lent cation induced structural transitions in telomeric DNAs:G-DNA folding intermediates. Biochemistry 1991;30:4460–72.[8] Gray RD, Chaires JB. Kinetics and mechanism of K+- andNa+-induced folding of models of human telomeric DNA intoG-quadruplex structures. Nucleic Acids Res 2008;36:4191–203.[9] Jixun D, Megan C, Chandanamali P, Roger AJ, Danzhou Y.Structure of the hybrid-2 type intramolecular human telomericG-quadruplex in K+ solution: insights into structure polymor-phism of the human telomeric sequence. Nucleic Acids Res2007;35:4927–40.[10] Yang D, Okamoto K. Structural insights into G-quadruplexes:towards new anticancer drugs. Future Med Chem2010;2:619–46.[11] Wang Y, Patel DJ. Solution structure of the human telomericrepeat D[Ag3(T2Ag3)3] G-tetraplex. Structure 1993;1:263–82.[12] Parkinson GN, Lee MP, Neidle S. Crystal structure of parallelquadruplexes from human telomeric DNA. Nature2002;417:876–80.[13] Li J, Correia JJ, Wang L, Trent JO, Chaires JB. Not so crystalclear: the structure of the human telomere G-quadruplex insolution differs from that present in a crystal. Nucleic AcidsRes 2005;33:4649–59.[14] Dai JX, Carver M, YangDZ. Polymorphism of human telomericquadruplex structures. Biochimie 2008;90:1172–83.[15] Buscaglia R, Miller MC, Dean WL, Gray RD, Lane AN, TrentJO, et al. Polyethylene glycol binding alters human telomereG-quadruplex structure by conformational selection. NucleicAcids Res 2013;41:7934–46.[16] Phan AT, Luu KN, Patel DJ. Different loop arrangements ofintramolecular human telomeric (3 + 1) G-quadruplexes inK+ solution. Nucleic Acids Res 2006;34:5715–9.[17] Zhang Z, Dai J, Veliath E, Jones RA, Yang D. Structure of atwo-G-tetrad intramolecular G-quadruplex formed by avariant human telomeric sequence in K+ solution: insightsinto the interconversion of human telomeric G-quadruplexstructures. Nucleic Acids Res 2010;38:1009–21.[18] Ambrus A, Chen D, Dai J, Bialis T, Jones RA, Yang D. Humantelomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands inpotassium solution. Nucleic Acids Res 2006;34:2723–35.[19] Gray RD, Trent JO, Chaires JB. Folding and unfoldingpathways of the human telomeric G-quadruplex. J Mol Biol2014;426. http://dx.doi.org/10.1016/j.jmb.2014.01.009.[20] Mashimo T, Yagi H, Sannohe Y, Rajendran A, Sugiyama H.Folding pathways of human telomeric type-1 and type-2 G-quadruplex structures. J Am Chem Soc 2010;132:14910–8.[21] Biffi G, Tannahill D, McCafferty J, Balasubramanian S.Quantitative visualization of DNA G-quadruplex structuresin human cells. Nat Chem 2013;5:182–6.[22] Karsisiotis AI, Hessari NM, Novellino E, Spada GP,Randazzo A, Webba da Silva M. Topological characteriza-tion of nucleic acid G-quadruplexes by UV absorption andcircular dichroism. Angew Chem Int Ed 2011;50:10645–8.[23] TorimuraM, Kurata S, Yamada K, Yokomaku T, Kamagata Y,Kanagawa T, et al. Fluorescence-quenching phenomenon byphotoinduced electron transfer between a fluorescent dyeand a nucleotide base. Anal Sci 2001;17:155–60.[24] Gray RD, Petraccone L, Buscaglia R, Chaires JB. 2-Aminopurine as a probe for quadruplex loop structures.Methods Mol Biol 2010;608:121–36.1628 Kinetics and Structures on the Molecular Path

W. David Wilson

Ananya Paul

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Kinetics and Structures on the Molecular Path to the Quadruplex Form of the Human Telomere

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