A Short GC-Rich Palindrome of Human Mannose Receptor Gene CodingRegion Displays a Conformational Switch

Aparna Bansal, Manoj Prasad, Kapil Roy, Shrikant KukretiNucleic Acids Research Laboratory, Department of Chemistry, University of Delhi (North Campus), Delhi 110007, India

Received 1 December 2011; accepted 4 June 2012

Published online 22 June 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22111

This article was originally published online as an accepted

preprint. The ‘‘PublishedOnline’’date corresponds to the preprint

version. You can request a copy of the preprint by emailing the

Biopolymers editorial office at biopolymers@wiley.com

INTRODUCTION

The discovery of polymorphic behavior of DNA was

contemporary to the discovery of DNA double helix.1

On the basis of amount of relative humidity present,

the DNA was categorized to A- and B-forms.

Although the most common or physiological form of

DNA is the B-form, a number of other polymorphic forms

subsequently been found are subclasses of A and B double

helices.2 These forms of DNA are interconvertible as a result

of the intricate interplay between the various factors like the

nature of the sequence, environmental solution conditions or

subject to interaction with different ligands, proteins, and so

forth. Among all the discussed factors, base composition and

base sequence have been considered as the most important

issue for the subsistence of the preferred conformation.3

Transition between A- and B-forms is reversible, cooperative,

and sequence specific.4 On the basis of X-ray crystallographic

analysis, A-DNA has been found to be more rigid than B-

DNA.5 Therefore, propensity of conversion of A-form to B-

form is less. However, there are certain reports in which A ?B transitions are discussed.6–9 Theoretical studies using mo-

lecular dynamic simulations have analyzed the A ? B con-

version in aqueous solution.10,11 Structural peculiarities of

such isomorphism might be of biological interest, and it is

therefore intriguing to explore the various factors that facili-

tate A ? B transition.

The advent of human genome has led to a manifold

increase of information in nucleotide sequence databases.

There are various types of repetitive sequences, including

microsatellites, dinucleotide repeats, triplet repeats, minisa-

tellites, and telomeres.12 Highly polymorphic from structural

point of view, such regions can assume several non-B-DNA

structures using non-Watson-Crick and Hoogsteen base pair-

ing.13,14 The most important aspect of DNA structural and

conformational variations is likely to be found in the

mechanics of molecular recognition and manipulation by

proteins, as they are required to manipulate the DNA struc-

ture to carry out their function.15–17

A Short GC-Rich Palindrome of Human Mannose Receptor Gene CodingRegion Displays a Conformational Switch

Additional Supporting Information may be found in the online version of this

article.Correspondence to: Shrikant Kukreti; e-mail: skukreti@chemistry.du.ac.in or

kukretishrikant@yahoo.com

ABSTRACT:

Conformational switching in DNA is fundamental to

biological processes. The structural status of a

palindromic GC-rich dodecamer DNA sequence, integral

part of human MRC2 coding region, and a related

sequence of opposite polarity from human FDX1 gene

were characterized and compared. UV-melting, circular

dichroism, and gel electrophoresis experiments

demonstrated the formation of intermolecular structures.

Although stability and molecularity of both the

oligomeric structures were found to be almost identical,

their secondary structures differed remarkably as A1

MRC2 sequence showed A-like and B-like DNA

conformation, whereas the A2 FDX1 sequence exhibited

only the A-like signatures. The study is relevant for

understanding structural polymorphism at genomic

locations depending on DNA sequence and solution

environment. # 2012 Wiley Periodicals, Inc. Biopolymers

97: 950–962, 2012.

Keywords: GC-rich palindromes; DNA structural

polymorphism; A ? B transition; MRC2; FDX1

Contract grant sponsor: University Grant Commission, New Delhi

Contract grant number: F.12-28/2002 (SR-1)

Contract grant sponsors: Delhi University and UGC

VVC 2012 Wiley Periodicals, Inc.

950 Biopolymers Volume 97 / Number 12

This work surfaced as a part of our ongoing interest in

studying structural polymorphism at palindromic/quasi-pal-

indromic repeats, homopurine–homopyrimidine segments

of genomic origin.8,9,18 Structural transitions between vari-

ous forms of DNA would have consequences in vivo, and a

thorough understanding of their physical and structural

properties is obligatory. Keeping in mind the polymorphic

nature of GC-rich regions and its possible biological roles,

we undertook biophysical studies of two self-complementary

deoxydodecanucleotide sequences d-GGCCGGCCGGCC

[A1] and d-CCGGCCGGCCGG [A2] differing in polarity

and their presence at distinct genomic locations. On going

through various databases, it has been found that both the

nucleotide sequences are present on specific genes across

human genome. The sequence A1 found on chromosome-17

is located in the coding region of mannose receptor (MRC2)

gene of human genome and is responsible for sugar binding,

Ca21 ion binding, and receptor activity. The sequence [A2],

which codes in part the FDX1 gene present on chromosome-

11, facilitates Fe2S2 cluster binding and electron carrier activ-

ity. These dodecamer sequences can also be found in many

mammalian genes at distinct coding and noncoding loca-

tions of various genes.19 Moreover, both the sequences also

possess cleavage sites GGCC, CCGG, GCCGGC, and

CGGCCG of the restriction endonucleases HaeIII, MspI/

HpaII, NgoM IV, and Eag I, respectively.

Herein, we report on the A ? B conformational switch

within the perfect duplex of sequence A1, contrarily, the

closely related sequence A2, differing just in the polarity (i.e.,

with 50-CC in place of 50-GG), completely lacks this struc-

tural behavior. Such studies might help in understanding the

conformational switching within DNA at the genomic poly-

morphic sites.

RESULTS

UV Thermal Melting Studies

First, the stability of the structural species formed by

(GGCC)3[A1] and (CCGG)3[A2] deoxydodecanucleotides

was investigated. For convenience sake, the sequence will be

named as [A1] and [A2] henceforth. Because of the self-com-

plementary nature of the sequences, they are expected to

form duplexes. For helix–coil transitions of the oligomeric

structures, the samples were subjected to UV thermal dena-

turation by monitoring at 272 nm. We did not find any dif-

ference between heating and cooling curves (melting and

annealing curves) of thermal denaturation profiles. No hys-

teresis was observed. Figure 1a shows the absorbance versus

temperature profiles of the palindromic MRC2 sequence

[A1] at 5 lM strand concentration in 20 mM sodium caco-

dylate buffer (pH 7.4) containing 0.1 mM EDTA and 0.1M

(Curve 1) or 1M NaCl (Curve 2). The melting curves dis-

played ‘‘nearly’’ sigmoidal monophasic transitions as a func-

tion of salt concentrations. Distinct melting temperatures

(Tms) calculated from the first derivative of the observed

thermal transition were found to be 73 and 778C at 0.1M and

1M NaCl, respectively. Likewise, the [A2] displaying mono-

phasic melting profile (Figure 1b) also showed salt concen-

tration dependence on its Tm. Surprisingly, the Tm values

showed a marginal increase even with 10-fold increase in salt

concentration.

Being palindromic, the sequences are likely to form

duplexes; however, prospects of hairpin formation cannot be

ruled out at this stage.20 Furthermore, to obtain the informa-

tion on the stability and molecularity of the structures,

oligomer concentration dependence on Tm was carried out.

It is apparent from Figures 1c and 1d that the melting pro-

files obtained at different oligomer concentrations are still

monophasic and demonstrate concentration dependence on

Tm values. The Tm of [A1], determined by first derivative

method, at 10 and 100 lM strand concentrations in the pres-

ence of 0.1M NaCl were found to be 748C (Curve 1) and

788C (Curve 2), respectively. Similarly, the thermal melting

experiments for [A2], carried out under identical solution

conditions (Figure 1d), also exhibited the dependence of Tmvalues on oligomer concentration by increasing it from 758C(Curve 1) to 788C (Curve 2). The concept of oligomer con-

centration dependence on Tm by intermolecular as well as

intramolecular structures has already been well illustrated in

the literature, and accordingly, the Tm of intramolecular

(unimolecular) structures is concentration independent,

whereas the Tm of intermolecular (bimolecular) structures is

concentration dependent.21 It is also important to mention

here that thermal denaturation of A1 was also carried out in

high salt (1M) at 10 and 50 lM oligomer concentrations, and

the DTm of two concentrations was found to be of 58C (data

not shown). On that account, our observation on the melting

profiles obtained for [A1] and [A2] manifest the intermolec-

ular (duplex) melting as their Tm show oligomer concentra-

tion dependence.

Additionally, we also recorded thermal difference spectra

(TDS) of both the sequences under study (Figure 2). Usually,

a TDS is obtained for a nucleic acid by simply recording the

ultraviolet absorbance spectra of the unfolded and folded

states at above and below its Tm. The TDS has a specific

shape that is unique for each type of nucleic acid structure.22

It was interesting to see that both the sequences displayed

characteristic TDS spectra corresponding to antiparallel GC-

rich duplexes.22 Using the potential of TDS method, we fur-

A ? B Transition in MRC2 951

Biopolymers

ther confirm that sequences A1 and A2 exist as duplex struc-

tures.

Furthermore, the molecularity of the identified intermo-

lecular structure formed by [A1] and [A2] sequences was

then detected by nondenaturating gel assays.

Nondenaturating Gel Electrophoresis

Nondenaturating gel electrophoresis was used to detect the

molecularity of the structural species, as demonstrated above

through thermal melting studies. It is important to mention

here that prior to performing gel assays in nondenaturating

FIGURE 1 Thermal denaturation profiles of (a and c) (GGCC)3[A1] and (b and d)

(CCGG)3[A2] in 20 mM sodium cacodylate (pH 7.4) and 0.1 mM EDTA. (a and b) Salt depend-

ence: (1) 0.1M NaCl and (2) 1M NaCl. (c and d) Oligomer concentration dependence: (1) 10

lM and (2) 100 lM.

FIGURE 2 Differential absorption spectra (TDS) of (a) (GGCC)3[A1] and (b) (CCGG)3[A2].

952 Bansal et al.

Biopolymers

conditions, the purity of the commercially made oligomers

was checked by running them on 20% polyacrylamide gel

containing 7M urea.

The [A1] and [A2], as well as the size markers, migrated

as single bands according to their size. The electrophoretic

mobility pattern of A1 and A2 sequences at varied strand

concentrations (10, 40, 70, and 100 lM) in 20 mM sodium

cacodylate (pH 7.4) containing 0.1M NaCl and 0.1 mM

EDTA is shown in Figure 3 (Lanes 2–5 for A1 and Lanes 6–9

for A2). The molecularity of [A1] and [A2] was predicted by

using two size markers 12-mer PAL (Lane 10) and a hetero-

duplex [12 base pairs (bp)] formed by mixing D1 and D2 in

1:1 ratio (Lane 1). The size marker PAL is a fully palindromic

12-nt long sequence, which under native gel conditions

moves as a 12-bp perfect duplex (it is a well-tested 12-bp

duplex marker used in the authors’ laboratory). Moreover,

the structural status of PAL was further supported by its

equivalent migration with a heteroduplex formed by mixing

two complementary dodecamer sequences (D1 and D2) in

equimolar ratio. The electrophoretograms of both [A1] and

[A2] under study were found to display the identical mobil-

ity patterns. A1 and A2 displayed single bands in a concen-

tration range of 10 ? 100 lM, migrating equivalent to 12-bp

heteroduplex (D1�D2) or PAL. As, like PAL, [A1] and [A2]

are also dodecamer palindromes, it is clear that the single

bands displayed separately by [A1] and [A2] correspond to a

bimolecular Watson-Crick duplex structure. It is worth men-

tioning that the gel data also ruled out our earlier speculation

about the possibility of formation of hairpin structures by

palindromic [A1] and [A2] sequences.

In addition, we also thought of the possibility of [A1] and

[A2] sequences existing as parallel stranded duplexes in the

gel. However, this option was ruled out on the basis of the

reports that parallel stranded duplexes (with reverse Watson-

crick base pairing) migrate faster relative to antiparallel

duplexes (with Watson-crick base pairing),23 and had they

been parallel-stranded duplex forms, they would migrate

faster than the D1�D2 heteroduplex or PAL. Hence, gel data

unambiguously demonstrate that MRC2 [A1] and FDX1

[A2] dodecamer sequences form bimolecular duplexes under

identical solution conditions.

Circular Dichroism Studies

CD spectroscopy, known to be extremely sensitive to poly-

morphism and small changes in global structure of nucleic

acids, was used for the secondary structure analysis, under

various solution conditions of the duplexes formed by [A1]

and [A2] sequences. Figure 4 shows the CD spectra of [A1]

and [A2], each at 10 lM concentration in 20 mM sodium

cacodylate buffer (pH 7.4), 0.1M NaCl, and 0.1 mM EDTA.

CD profile of A1 displays a negative peak at 242 nm, and the

two positive peaks near 263 and 286 nm and the other

sequence with opposite polarity [A2] showed negative peak

at 245 nm followed by a single positive peak at 263 nm. A

typical B-DNA structure is characterized by a negative CD

band at 255 nm followed by a positive peak of equal ampli-

FIGURE 3 Native PAGE mobility pattern (20%) of the oligonu-

cleotide sequences. Lane 1: D1lD2(1:1); Lanes 2–5: (GGCC)3[A1];

Lanes 6–9: (CCGG)3[A2] at 10, 40, 70, and 100 lM; Lane 10: PAL in

20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and 0.1M NaCl.

FIGURE 4 CD spectra of (GGCC)3[A1] (n) and (CCGG)3[A2]

(*) at 10 lM strand concentration in 20 mM sodium cacodylate

(pH 7.4), 0.1 mM EDTA, and 0.1M NaCl.

A ? B Transition in MRC2 953

Biopolymers

tude at 285 nm, whereas A-form displays the negative band

at 240 nm and positive centered at 265 nm.24 The analysis of

Figure 4 suggests characteristics of both the A- and B-like

DNA structures adopted by [A1]. Interestingly, CD charac-

teristics of [A2] exhibited only the A-like DNA features. The

prospects of existence of a duplex structure possessing A-

and B-like features, as revealed from CD studies, made this

study intriguing for further investigation. On the lines dis-

cussed above in melting and gel studies, CD profiles of [A1]

and [A2] were also scanned as a function of oligomer and

salt concentrations. Figures 5a and 5b show the CD profiles

of [A1] and [A2] at various concentrations (10–35 lM) in

0.1M NaCl. The [A2] as shown in Figure 5b displayed a posi-

tive peak centered about 263 nm and negative peaks at 245

and 210 nm at the lowest concentration used (10 lM), which

persisted up to 35 lM, followed by a gradual increase in the

positive and negative CD amplitudes. Thus, CD characteris-

tics mark the presence of A-like DNA structure of [A2],

whereas for [A1], as shown in Figure 5a, of the well-defined

negative peak around 210 nm and two positive peaks at 263

and 286 nm at 10 lM oligomer concentration, only the lon-

ger wavelength positive peak survived at higher concentra-

tion (35 lM). As 280–285 nm positive peak in CD is attrib-

uted to the B-type DNA conformation, oligomer concentra-

tion dependence on CD signals clearly reflect the A ? B

transition in [A1] sequence.

We next investigated the effect of salt on the CD spectra

(Figure 6a) at low oligomer concentration (10 lM), where

the duplex exhibited A- and B-like features, and at high

oligomer concentration (30 lM) showing only the B-like fea-

tures (Figure 6b). It is apparent that except a marginal

increase in long wavelength CD amplitude of [A1], as shown

in Figures 6a and 6b, at 10 and 30 lM oligomer, respectively,

no shift was observed in the signal peaks as a function of salt

concentration. Likewise, the sequence [A2] (Figure 5b),

which showed only the features of A-like DNA, displayed

only a subsidiary increase in CD amplitude at 263 nm (data

not shown). This marginal increase in CD amplitude can be

FIGURE 5 CD spectra of (a) (GGCC)3[A1] and (b) (CCGG)3[A2] in 20 mM sodium cacodyl-

ate (pH 7.4), 0.1 mM EDTA, and 0.1M NaCl at different oligomer strand concentration.

FIGURE 6 CD spectra of (GGCC)3[A1] at (a) 10 lM and (b) 30 lM strand concentration in

20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and 0.1M (*) and 1M (n) NaCl.

954 Bansal et al.

Biopolymers

attributed to the stabilization of duplex secondary structures.

Thus, no significant change in CD profiles of both [A1] and

[A2] with increase in the salt concentration from 0.1M to 1M

indicate that the subsistence of [A1] and [A2] conformation

is not predisposed within the studied range of NaCl concen-

tration.

To confirm the presence of true A-DNA form, anticipated

above in [A1] and [A2] sequences, the use of a conventional

agent trifluoroethanol (TFE) was made to induce B ? A

transition in the aforementioned sequences. Figures 7a and

7b display CD profiles of [A1] and [A2] at 10 lM strand con-

centration under the identical buffer conditions used in Fig-

ures 4a and 4b with the addition of 60% TFE. As expected,

the amplitude of 265 nm positive CD peak, attributed to the

A-form DNA, increased in the presence of TFE. We took this

observation as a diagnostic for the presence of A-like features

in MRC2 (A1) and FDX1 (A2) sequences.

Furthermore, in the presence of 60% TFE, the spectrum

shows increase in magnitude of both peaks at 265 and 286

nm. For the moment, the increase in the CD magnitude at

286 nm remains inexplicable; however, when compared,

there is a significant increase at the 265 nm peak correspond-

ing to significant induction of A-like structure. As higher

TFE concentrations led to sample condensation, we used

another conventional agent ethanol to show CD spectrum of

the true A-form. Remarkably, the various CD spectra

recorded as a function of ethanol concentration showed a

complete cooperative ethanol-induced transition for [A1]

(Figure 8). However, in case of [A2] possessing A-like fea-

tures, with each addition of ethanol, an increase in 265-nm

CD amplitude was observed (data not shown), similar to

that noticed at TFE addition (Figure 7b). This observation

further reinforced our conclusion that the A ? B transition

shown for sequence MRC2 (A1) could be simple isomeriza-

tion of the palindromic duplex between two conformations.

Our gel data (discussed above) have conclusively ruled

out the possibility of [A1] and [A2] existing in hairpin

duplex equilibria. However, in the light of our earlier study8

on quasi-palindromic GnXCn sequences (X 5 A,G,C,T),

which existed in hairpin-duplex equilibria and possessed ele-

ments of A- and B-type DNA conformations, we still per-

formed additional independent experiments to test this pos-

sibility with [A1] and [A2] sequences. Hairpin to duplex

transition is known to be very cooperative for sequences of

the (CnGn)m and (GnCn)m type and is accompanied by dis-

tinct changes in their anomalous structures reflected in CD

spectra.8,9 Furthermore, because of the cooperativity of the

hairpin-duplex transition, the populations of the particular

FIGURE 7 CD spectra of (a) (GGCC)3[A1] and (b) (CCGG)3[A2] at 10 lM strand concentra-

tion in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and absence (n) and presence (*)

of trifluoroethanol (TFE).

FIGURE 8 CD spectra of (GGCC)3[A1] at 10 lM strand concen-

tration in 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and

50% (n), 60% (*), 65% (*), 70% (~), and 80% (^) of EtOH.

A ? B Transition in MRC2 955

Biopolymers

conformers strongly depend on sample preparation. Keeping

these facts in mind, CD spectra of the samples were prepared

by two different methods. It is a fact that rapid removal of

denaturating conditions as quickly cooling a heat-denatured

DNA sample (cold shock state) results in the collapse of sin-

gle strands into compact unimolecular structures (hairpins

or random coils) stabilized by intramolecular hydrogen

bonding between complementary segments. Rapid unimolec-

ular processes can be 100 times faster than the corresponding

bimolecular pairing process. As a manifestation of this phe-

nomenon and to see if [A1] and [A2] exhibit any hairpin-

duplex equilibrium, we compared the CD spectra of [A1]

(standard sample preparation) with melted [A1] (quickly

quenched to 08C on ice). CD spectra of both the samples

([A1] at 10 lM concentration), as shown in Figure 9a,

showed identical secondary structures having A-like DNA

feature. Likewise, at the concentration (30 lM) at which

[A1] is shown to adopt B-like structure (Figure 9b), the CD

spectra of two different samples were again found to be

almost identical. This clearly confirms that [A1] does not ex-

hibit hairpin duplex equilibrium. Similarly, the CD spectra of

[A2] recorded with above-mentioned sample preparations

were found to be identical (data not shown), indicating that

like [A1], [A2] also forms a single bimolecular duplex spe-

cies. Therefore, the switch from a mixed A and B conforma-

tion to a B-type conformation at high oligonucleotide con-

centration, exhibited by [A1], is not a transition between

hairpin and duplex, but intrinsic conformational changes in

the duplex.

Furthermore, to check whether there is any effect of

neighboring bases on the sequence polarity of [A1] and [A2],

a control experiment was done, accordingly extended version

of both A1 and A2 sequences (named A1–20 and A2–20)

were designed. [A1] and [A2] were flanked by four bases at

50- and 30-ends. To make the study more relevant, we did not

select random bases as flanking sequences, but all the eight

bases were derived from the gene sequence of [A1] and [A2],

that is 1 8 bases of A1 in MRC2 and A2 in FDX1 genes. The

flanked (extended) versions of [A1] and [A2] sequences are

as follows:

� 50-GCC CGG CCG GCC GGC CCC CG-30 A1–20

� 50-CGG CCC GGC CGG CCG GTG GC-30 A2–20

CD spectra of A1–20 and A2–20 were recorded (data not

shown) under same solution conditions used for CD experi-

ments on [A1] and [A2] (Figure 4). It is interesting to find

that A1–20 displayed CD spectrum quite identical to CD

spectrum of [A1] with two positive peaks corresponding to

the CD signatures of A- and B-like DNA conformation. CD

spectrum of A2–20 also showed the predominant A-like sig-

natures along with small positive hump exhibiting elemen-

tary B-like features. The experiment suggests that the sequen-

ces [A1] and [A2] from the MRC2 and FDX1 genes almost

retain their conformational characteristics even when flanked

by nucleotides.

Going a step further, for testing the sequence effect of

[A1] and [A2], we mutated the sequences [A1] and [A2] by

deleting one G from each GGCC unit of [A1], making it

GCC and similarly C from each CCGG unit making it CGG

of [A2]. However, the length of the sequences was kept same

as of [A1] and [A2] by increasing one trinucleotide unit [i.e.,

new dodecamers (GCC)4 and (CGG)4 named A4 and A3,

respectively]. The sequences are as follows:

� 50-CGG CGG CGG CGG-30 A3

� 50-GCC GCC GCC GCC-30 A4

CD spectra recorded (Supporting Information Figure S1)

under the same solution conditions, as applied in Figure 3,

show that the A3 and A4 adopt pure A-like and B-like forms

of DNA characterized by �260 nm positive/245 nm negative

FIGURE 9 CD spectra of (GGCC)3[A1] at (a) 10 lM and (b) 30 lM strand concentration in

20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and 0.1M NaCl before (n) and after (*)

denaturation.

956 Bansal et al.

Biopolymers

and �285 nm positive/255 nm negative CD bands, respec-

tively.24 It is worth mentioning that the lack of consecutive

CC and GG steps in A3 and A4 results in only one type of

CD signatures corresponding to A- or B-like DNA conforma-

tions. Furthermore, to confirm A ? B transition, the repre-

sentative spectra of A3 and A4 have been overlaid with

oligomer concentration dependence CD spectra (Figure 4a)

of A1 (Supporting Information Figure S4). The role of CC

and GG steps has already been discussed in the text.25–29

Thus, the control experiments support our conclusion

that it is the intrinsic property of A1 (GGCCGGCCGGCC)

and A2 (CCGGCCGGCCGG) sequences, which in spite of,

identical molecularity (duplexes) and thermal stability,

exhibited marked difference in their secondary structures.

The A1 (MRC2) sequence showed A- and B-like DNA con-

formation, whereas the A2 (FDX1) exhibited predominantly

the A-like signatures.

Nonexistence of Multistranded (Triplets and

Quartets) Structures

The formation of multistranded structures is a well-docu-

mented phenomenon for short oligomeric sequences with

guanine tracts.30 It is worth mentioning that the CD spec-

trum of [A2] is close to a parallel G-quadruplex signature.

Similarly, the [A1] could easily represent a mixture of parallel

and antiparallel G-quadruplex species and B-DNA. We argue

that because of the self-complementary nature of [A1] and

[A2] and the absence of a contiguous G- or C-stretches in

their sequences (a pre-requisite of G- and C-quartet forma-

tion), the possibility of the formation of G-quadruplex or i-

motif structures by [A1] and [A2] can be ruled out. More-

over, the CD spectrum of [A2], which shows the expected

CD band at 260–265 nm of the parallel G-quadruplex, lacks

a positive band at 210 nm, which is also characteristic of the

quadruplex.

Mergny et al.31 have shown that G-quartet formation can

be followed by UV spectroscopy. However, we performed UV

thermal melting on both the [A1] and [A2] palindromes. As

expected, the UV-melting profiles of [A1] and [A2] moni-

tored at 295 nm did not show an inverse sigmoidal curve

(data not shown), a feature that is a diagnostic of G-quadru-

plexes.31 Thus, the presence of G- or C-quadruplexes was

excluded. Furthermore, a careful observation of [A1] and

[A2] rules out the possibility of the formation of triplexes by

these sequences, as such structures require a pure oligopyri-

midine–oligopurine stretch in the target DNA. Interruption

in the oligopyrimidine–oligopurine target sequences by one

or more inverted purine–pyrimidine base pairs leads to dra-

matic decrease in triplex stability.32 Additionally, the gel pic-

tures (Figure 3) did not show any unidentified retarded

bands, which would confirm the presence of higher order

structures other than the intermolecular duplexes.

DISCUSSIONIt has become increasingly apparent in recent years that DNA

polymorphism is a multidimensional phenomenon. The

double-helical structure of DNA is remarkably stable. It is an

association of two antiparallel strands, which stabilize the

double helix through its intrastrand as well as interstrand

properties. They are found to be dominating over each other

depending on the surrounding conditions. The B- and A-

forms of DNA differ from each other in several characteris-

tics such as base-stacking geometry, backbone geometry, and

hydration.2

Present [A1] GC-deoxydodecamer of genomic origin is

the first example of a 50-purine dinucleotide, short perfect

duplex, which exhibited A ? B transition, as a function of

oligomer concentration. Both UV thermal melting and the

gel assays provide a clear picture about the bimolecular status

and the stability of duplexes formed by [A1] and [A2]

sequences. However, their distinctive conformational status

is important to be discussed. We have previously shown that

an oligomeric structure formed by an imperfect palindrome,

existing in hairpin-duplex equilibrium and possessing ele-

ments of A- and B-type DNA conformations, can switch over

to a single predominant structural species as a function of

oligomer concentration. This hairpin ? duplex switch was

interpreted in terms of A ? B transition, where unimolecu-

lar hairpin species adopts A-form, whereas B-form is the pre-

ferred conformational state bimolecular duplex. The results

indicated possibility of an architectural switching between

linear duplex to cruciform structure associated with the

structural polymorphism at the quasi-palindromic region of

b-globin gene LCR.8,9

Working on similar lines, we showed here a novel polymor-

phism where with mere change in the oligonucleotide concen-

tration, a perfect Watson-Crick duplex displaying both A- and

B-like features can undergo A ? B switch. Prevalence of the

two conformations in a perfect Watson-Crick duplex structure

draws attention toward the role of oligonucleotide concentra-

tion factor, stabilizing B- and A-like forms in sequences [A1]

and [A2], respectively. It is well known that in B-DNA, the

stacking is largely limited to interactions between bases on

same polynucleotide chain, whereas in A-form, stacking also

involves bases belonging to two different chains, that is, both

interstrand and intrastrand stacking.2 Calladine and Drew25

elegantly proposed that transition is driven by base pair stack-

ing resulting in change in the sugar conformation. Mounting

A ? B Transition in MRC2 957

Biopolymers

evidences suggest about the role of sequence to be important

in exhibiting conformational polymorphism in DNA.26–28

Kypr and coworkers showed that aqueous duplex of

(GGGGCCCC) is characterized by two strong positive bands at

260 and 285 nm marking the presence of both A- and B-like

DNA features. In this duplex, half of the C bases are assumed

to be stacked in B-like fashion and other G bases in A-form,

but within the B-DNA framework. NMR spectroscopy showed

that the anomalous structure has an A-like stacking of bases

but a B-type sugar puckering. Except some A-like features, the

duplex does not deviate from B-type DNA helix. Therefore,

based on the reports, it is conceivable to assume that the 267

and 285 nm positive CD signals for [A1] correspond to A-like

and B-like DNA conformation contributed by GG, GC, and

CC stacks. It is conceivable that because of the presence of GG

steps, the single B-DNA duplex structure of (GGCC)3 has also

some A-like features. Similarly, the octamer d(CCCCGGGG)

and other d(CnGn) fragments of DNA provide CD spectra,

which suggest that the base pairs are stacked in an A-like fash-

ion even in aqueous solution. However, C4G4 providing the

huge positive 260 nm CD signal (but lacking the negative one

at 210 nm) is the A-form as it cooperatively transforms into

the very A structure in TFE. NMR spectroscopy showed a B-

type puckering of the deoxyribose sugar ring. Hence, a combi-

nation of the information provided by CD spectroscopy and

NMR suggests an unprecedented double helix of DNA in

which A-like base stacking is combined with B-type sugar

puckering. Moreover, the effect of G-tract length and flanking

sequences on the solution conformation of DNA has also been

studied by Lindqvist and Graslund.29 Accordingly, using CD

and FTIR spectroscopy, they showed the predisposition of

A-type conformation in sequences (CATGGGCCCATG)2,

(AGGGGCCCCT)2, and d(TGGGGCCCCA)2 depending on

the length of G tract as well as the sequence context.

With our MRC2 sequence [A1], the amplitude and posi-

tion of both the CD peaks demonstrate the signatures of

both the A- and B-conformations. The factors like hydration

spine of DNA molecule, intermolecular bonds, and stacking

interactions play a vital role in controlling the DNA confor-

mation.2,33 The transition from A-form to B-form in A1 with

the increase in oligomer concentration might be attributed

to some changes in the solution environment. There are

marked differences in the stacking patterns in A- and B-

DNA. In B-DNA, only intrastrand stacking is observed: base

overlap in Pu–Py and Pu–Pu steps is good and contrasts with

the poor stacking even at Py–Py and Py–Pu. In A-DNA, there

is almost non-Py–Py stacking; however, significant inter-

stacking overlap of purines occurs at Py–Pu steps.2 We specu-

late that molecular interactions at high oligomer concentra-

tion in case of [A1] (consisting of both A and B conforma-

tions) might result in the weakening of intrastrand stacking

(favoring A-form of DNA), which consequently might

change the sugar conformation resulting in change in the

conformation. The observed A ? B switch could be a conse-

quence of such intrinsic distortion in base pair structures.

Moreover, the thermodynamic barrier between A- and B-

forms is not very large.34

While discussing the fact that A-form duplexes have cross-

strand stacks between guanine bases at 50C-G-30 steps in the

sequence, the possibility of structures slipped from perfect

duplexes to make extended sets of paired molecules cannot be

ignored. Accordingly, sequence [A1] can have cross-strand

stacks that bridge gaps at the ends of strands, stabilizing the

top structure in the A-form, whereas [A2] cannot have cross-

strand stacks extending across the gaps. Accordingly, forma-

tion of slipped / extended structures should be more favorable

with increasing concentration of [A1], than [A2]. There is

another distinguishable slipped state where the bottom row is

slipped four bases to the right; cross-strand G/G stacks can still

occur for [A1] but not for [A2]. Slipping four bases to the left

makes perfect duplexes, which also offer the possibility of G/G

cross-strand stacks if the double helix continues across the ter-

mini of the duplexes (Supporting Information Figure S5).

However, our primary argument against such slipped struc-

tures is the native gel experiments in Figure 3. These show

structures consistent with bimolecular structures even at high

oligomer concentrations rather than the multimolecular spe-

cies speculated above.

An X-ray study, made more than a decade ago,35 showed

that the phosphate of CpG step in d(GCGCGCGCGC) is

involved in hydrogen bonding to the water ring network,

whereas that of GpC step is not, suggesting why the GpC

phosphate exhibited greater flexibility than the CpG step. We

predict a similar situation in our studies. A closer inspection

of our sequences (GGCCGGCCGGCC) [A1] and

(CCGGCCGGCCGG) [A2] reveals the presence of three GpC

steps in [A1], providing more flexibility to the so-formed dou-

ble helix than the duplex formed by [A2] containing two GpC

steps. This could be one of the possible reasons why MRC2

sequence [A1] shows elements of A- and B-forms of DNA.

The status of a self-complementary octamer

d(GGCCGGCC), which is basically a truncated version of

our A1 sequence d(GGCCGGCCGGCC), has been investi-

gated by NMR, concluding that this octanucleotide adopts a

B-type DNA helix in the solution.36 Interestingly, by X-ray

studies, the same octamer d(GGCCGGCC) was shown to

exist in A-form.37 The 50-terminal base type Pu/Py has an

influence on the stacking and handedness of the short alter-

nating oligonucleotide.38 NMR and optical spectroscopy

techniques have demonstrated that the conformation of a

958 Bansal et al.

Biopolymers

specific oligonucleotide can significantly differ in solution

and crystal.36,39

It is important to mention here that though both A- and

B-forms of DNA have been largely studied by X-ray and

NMR methods, the reason for the preference for one confor-

mation over the other is still unclear. It is tedious to detect

the conformations existing in solution at micromolar con-

centrations. In an independent study, we are finding it diffi-

cult to solve a NMR structure of a DNA hairpin conforma-

tion, which is demonstrated to exist only in the range of 10–

40 lM oligomer concentration, above which it converts into

a bulge duplex form.8,9 Similarly, in this work, the A-form of

the MRC2 (A1) sequence was only detected below 30 lMoligomer concentration, and hence, further structural details

are required to uncover the reason for this sequence specific

structural polymorphism.

It has already been established that the junction between a

left-handed Z-DNA sequence and a right-handed B-DNA

sequence is about 3 bp and does not affect the ability of bases

to pair on either side of this region.40 A similar case was noted

for a junction between A-DNA and B-DNA in the oligomer

dGn(rC11dC16). The hybrid end of this molecule dG.rC

sequence assumes the A-conformation, whereas the DNA

duplex end with dG.dC sequence assumes B-conformation.

The junction between regions is localized to one base pair, and

there is complete base pairing and base stacking on both sides

of the junction.41 Studies indicated that such block polymers

are linear duplexes with two adjoining conformations with

minimal disruption of the helix at the junction of two confor-

mations. Thus, the promotion of the A ? B transition of syn-

thetic DNA oligomers by changing solution conditions may be

a good model for the conformational change in vivo.

A certain repeat pattern of naturally occurring DNA

sequences could have intrinsic as well as environmentally

induced structural polymorphism. Our study also highlights

the fact that the well-celebrated, canonical B-form helix is

not likely to exist in vivo because the actual shape of a region

of DNA will depend on its base composition, local sequence

environment on either side of the region, and the composi-

tion of the cellular milieu.13 The local conformation of DNA

duplex may be important in interactions between nucleic

acids and protein or drugs.

Biological Significance

It is interesting to consider that structurally altered DNA seg-

ments might serve as a regulatory signal for specific protein/

ligand binding event. As one segment of DNA can adopt

sequence-specific multiple conformations, different proteins,

each with a specific function, may recognize the various con-

formers and hence suggest multiple biological effects. How-

ever, a clear-cut example of such a relation is not yet avail-

able. The structural polymorphism of DNA can be predicted

from minor distortion to major deviation from the B-DNA

structure. The literature is rich in studies showing that crea-

tion of different forms of DNA depends on the underlying

nucleotide sequence and is influenced by the environment

and overall DNA topology. Proteins may read and recognize

sequence-dependent structural features of DNA.

The behavior of DNA in vivo is very complex. Extensive

in vitro studies on DNA polymorphism with recombinant

plasmids, short synthetic oligonucleotide sequences, restric-

tion fragments, and so forth have variably exploded our

knowledge about different DNA structures and conforma-

tions and relate to the in vivo existence and functions of cer-

tain structures.42 Of the various polymorphic forms, classical

B-DNA structure could be one of the conformations adopted

in vivo. TFIIIA conformational features recognize an A-

form,43 which is thought to be more readily available for ini-

tiation of transcription.44

Our finding of a structural polymorphism, manifested in

a switch between A ? B form, exhibited intrinsically by a

short GGCCGGCCGGCC dinucleotide repeat of genomic or-

igin, suggesting the possibility of recognition of a specific

form of DNA by proteins. To find the prevalence of the two

GC-rich palindromic dodecamer sequences [A1] and [A2]

under study, we searched the complete human genome data-

base (May 2008 release). Both sequences were found at sev-

eral coding and noncoding locations in human genome. For

instance, [A1] is found in the coding region of mannose re-

ceptor C type 2 (MRC2) gene at chromosome 17. Similarly

for [A2], using the available database, various genes of differ-

ent chromosomes were screened and the positions of this tar-

get were marked. Interestingly, in the case of MRC2 gene,

both the sequences were found to be located in the same

region, except that the position of [A2] is shifted by two

bases upstream. Furthermore, the database analysis for the

coding sequence of MRC2 revealed that although both the

target sequences [A1] and [A2] are present in the coding

region, cytosine at 50-end of [A2] does not participate with

rest of the sequence to code for amino acids, whereas [A1]

accounts for coding the tetrapeptide Gly-Arg-Prol-Ala. The

contribution of [A1] sequence in coding for a region of pro-

tein enhances its implication in the biological processes.

In sequence-specific recognition process, a protein may

sense the overall 3D structure of DNA and negotiate the

potential sites of hydrogen bonding available at major and

minor grooves. At this stage, although we do not have any

compelling evidence for a direct biological role of the

sequences under study, the occurrence of DNA binding sites

for various transcription factors (TFs), in the vicinity of A1

A ? B Transition in MRC2 959

Biopolymers

and A2 palindromes on MRC2 and FDX1 genes, respectively

(Supporting Information Figures S2 and S3), signifies that

TFs might show specificity or selectivity for structural fea-

tures rather than for a particular sequence and thus would

preferentially bind to one conformation of DNA. A variety of

biological processes require local separation of the two

strands of DNA. It is possible that GC-rich regions, which

require more free energy to separate, than do the AT region,

at times may change intrinsically to other isomorphic confor-

mation when strand separation is not required for protein

recognition. In an elegant study, Dickerson and coworkers45

reported on a transition from B- to A-form DNA at Nar LTF

binding site. The study concludes that DNA is recognized by

the concerted effect of solvation, van der Waals forces, and

inherent DNA deformability rather than determined primar-

ily by major groove hydrogen bonding.

A minor groove binding tract (MGBT) structural element

of HIV-1 transcriptase is important for both replication fra-

meshift fidelity and processivity. Interestingly, the MGBT

interactions occur in the DNA minor groove, where the DNA

undergoes a structural transition from A-form to B-from

DNA.46 The transition from the B-form to A-form of DNA is

also essential for biological functions as shown by the exis-

tence of A-form in many protein–DNA complexes.47 It has

been shown using X-ray crystallographic studies that DNase

I can bind to both an A-DNA sequence and B-DNA sequence

but can cleave only B-DNA sequence.48,49 Thus, various con-

formations of DNA (A, B, Z, etc.) would affect its local topol-

ogy that would either enhance or limit protein recognition.

Consequently, the regulation occurs not only at protein level

but also at that of DNA conformation.50–54

However, there are always questions whether the unusual

secondary structures of a short DNA segment of the comple-

mentary/noncomplementary nature is capable of adopting in

vitro may also be found in vivo. The intensity of recent

research on DNA structural polymorphism attests to the

awareness that structures other than canonical B-form shown

to be formed by short oligonucleotides of genomic sequences

may play important biological roles. Simple motifs in DNA

secondary structures may be important for protein recogni-

tion.55,56 Although our studies are far from the in vivo pic-

ture, biophysical structural characterization of the model

DNA fragments (oligonucleotides) in solution render ap-

proximate, overall structural information closely associated

with their possible biological status.

CONCLUSIONUsing a combination of optical melting, gel electrophoresis,

and circular dichroism studies, we conclude that the aqueous

solution of the studied dodecamer oligonucleotide sequences

[A1] and [A2] found at the coding regions of MRC2 and

FDX1 genes, respectively, associate into a perfect duplex hav-

ing varied conformations. The secondary structure analysis

by CD revealed that A2 exhibited only the A-like form of

DNA and did not show any dependence on oligomer concen-

tration, whereas [A1] displayed signatures of both A- and B-

like DNA conformation at low oligomer concentration (10

lM), which on further increase of concentration up to 30

lM showed a complete A ? B transition. This conforma-

tional variability shown by MRC2 [A1] gene segments

reflects the contribution of sequence context, base stacking

properties, and backbone geometry in facilitating structural

polymorphism. We believe that such findings emphasize the

importance of careful understanding of the molecular

switching and a better analysis of the sequence-dependent

variations of the DNA structure. Hence, this finding presents

a paradigm for the understanding of conformational diver-

gence in a perfect duplex as a function of oligomer concen-

tration. These sequence structural modulations could be the

features essential for protein–nucleic acids recognition in

terms of TFs binding.

MATERIALS AND METHODSThe oligonucleotides used were purchased from Bio Basic (Canada) in

the lyophilized powder form on 1-lM scale with PAGE purification.

They were stored at 2208C. The concentration of the oligonucleotides

was determined spectrophotometrically by using the extinction coeffi-

cient (e) calculated by nearest neighbor method and measuring the ab-

sorbance at 260 nm. The e value used for d-GGCCGGCCGGCC (A1),

d-CCGGCCGGCCGG (A2), d-GCCCGGCCGGCCGGCCCCCG

(A1–20), d-CGGCCCGGCCGGCCGGTGGC (A2–20), d-CGGCG

GCGGCGG (A3), d-GCCGCCGCCGCC (A4), d-CTCTTTTCCTTC

(D1), d–GAAGGAAAAGAG (D2), and d-CTTGAGCTCAAG (PAL)

were 102,900, 103,300, 166,200, 172,300, 108,500, 96,500, 92,900,

139,500, and 113,700 M21 cm21, respectively. Stock solutions of the

oligomers were prepared by directly dissolving the lyophilized powder

in MilliQ water. Equimolar mixture of D1 and D2 oligonucleotides

resulting into a 12-bp duplex and palindromic PAL were used as size

markers in gel assays.

Nondenaturating Gel ElectrophoresisFor performing gel experiments, native 20% polyacrylamide gel was

used. The samples were heat treated at 958C for 5 min followed by

slow cooling to room temperature and kept overnight at the same

temperature. The oligonucleotides were incubated at 48C for 1 h

before loading into the gel. Both the gel and samples contained 20

mM sodium cacodylate, 0.1 mM EDTA, and 100 mM NaCl, whereas

running buffer contained 13 TBE and same amount of EDTA and

salt. The gels were run at a constant voltage of 65 V in cold room

with maintained temperature of approximately 4–58C. The trackingdye used was orange G mixed with glycerol. After electrophoresis,

960 Bansal et al.

Biopolymers

the gels were stained with ‘‘stains all’’ (Sigma) solution prepared in

formamide and finally visualized under white light and photo-

graphed by AlphalmagerTM 2200 (Alpha Infotech Corp.).

UV Thermal DenaturationThermal denaturation experiments (Tm) were performed on

UV-1650PC Shimadzu UV–Vis spectrophotometer [equipped with a

Peltier thermoprogrammer, TMSPC-8 (E) 2 200]. Samples were pre-

pared and were incubated overnight at 48C. UV-melting curves were

acquired at 272 nm by heating the samples from 20 to 1008C with the

heating rate of 0.58C min21 using the Stoppered quartz cells of 10

mm path length with volume capacity of 110 lL. We did not find any

difference between heating and cooling curves (melting and annealing

curves) of thermal denaturation profiles. No hysteresis was observed.

For studying the high oligomer concentrations, the quartz cells of 1

mm path length with volume capacity of 35 lL were used.It is important to mention that Tms obtained as a function of salt

concentration as well as oligomer concentration differed by only 2 to

48C. To examine the significance of these differences, the reproduci-

bility was checked by conducting replicate independent experiments

and by reporting the standard deviations of the Tms and P-values for

the differences. The experiments were conducted more than three

times. The Tm values showing salt concentration/oligomer concentra-

tion dependence were reproduced, that is, Tm values of small

difference persisted. The standard deviation of the Tms for sequence

A1(d-GGCC)3 at 0.1M Na1 and 1M Na1 was found to be 0.5 for

both the concentrations, and at 10 and 100 lM oligomer, it was

calculated as 1 and 0.974, respectively. The P-values measured for Tms

of different salt, as well as oligomer concentration, were 0.0000144

and 0.000855, respectively. As the calculated values for standard

deviation and P-value are substantially small, it shows that the Tmmeasurements at different salt and oligomer concentrations are con-

siderable. Similarly, the standard deviation and P-values of Tms of A2

(d-CCGG)3 were also found to be significant. As both the sequences

carry the same GC content and form bimolecular duplexes, it is con-

ceivable to assume identical thermal stability for them.

Circular DichroismFor secondary structural analysis, CD spectroscopy was used. The

CD measurements were performed on JASCO-815 spectrophotome-

ter interfaced with an IBM PC compatible computer, calibrated

with D-camphor sulfonic acid. The samples were prepared and incu-

bated overnight at 48C. CD spectra were collected as an average of

four multiple scans between the ranges of 205–350 nm at the scan-

ning rate of 100 nm min21 at 208C using quartz cells of 10 mm

path length with volume capacity of 1 mL. Data were collected in

terms of milli degrees versus wavelength.

Database SearchingThe human genome sequence used to determine the distribution of

the two fully palindromic sequences was obtained from the NCBI

website (http://www.ncbi.nlm.nih.gov/genomes/; May 2008 release).

From the available data, information about gene locations and

intron/exon positions was extracted chromosome wise. Further-

more, the transcription binding sites (for 22000:1500) were

located using TFM explorer program.

The authors thank Dr. Ritushree Kukreti and Ms. Harpreet Kaur

(IGIB, CSIR, Delhi) for their kind help in genome database search.

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Reviewing Editor: Sarah A. Woodson

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