Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures.

8 MB·English

by  Hunt, Rebecca A.

Published online 25 January 2011 Nucleic Acids Research, 2011, Vol. 39, No. 10 4265–4274
doi:10.1093/nar/gkq1362
Induced topological changes in DNA
complexes: influence of DNA sequences
and small molecule structures
Rebecca A. Hunt, Manoj Munde, Arvind Kumar, Mohamed A. Ismail,
Abdelbasset A. Farahat, Reem K. Arafa, Martial Say, Adalgisa Batista-Parra,
Denise Tevis, David W. Boykin and W. David Wilson*
Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
Received November 5, 2010; Revised and Accepted December 30, 2010
ABSTRACT INTRODUCTION
Heterocyclic diamidines are compounds with anti- Heterocyclic diamidines, and related compounds, with an
parasitic properties that target the minor groove of appropriatestructurecanbindstronglytotheDNAminor
groove and exhibit important biological effects including
kinetoplast DNA. The mechanism of action of these
clinical antiparasitic activity (1–4). Recent results show
compounds is unknown, but topological changes
that the compounds can compete with transcription
to DNA structures are likely to be involved. In this
factor binding in both the minor and major grooves
study, we have developed a polyacrylamide gel
(5,6). The compounds can also target AT-rich sequences
electrophoresis-based screening method to deter-
in mitochondrial kinetoplast minicircle DNA (kDNA) in
mine topological effects of heterocyclic diamidines eukaryotic parasites that cause human disease (1–4), and
onfourminorgroovetargetsequences:AAAAA,TTT they have entered Phase III clinical trials against Human
AA, AAATT and ATATA. The AAAAA and AAATT African Sleeping Sickness (4). The kinetoplast is
sequences have the largest intrinsic bend, composed of maxicircles that contain the mitochondrial
whereas the TTTAA and ATATA sequences are genomic DNA and thousands of 1000–2000bp catenated
relatively straight. The changes caused by binding DNAminicirclesthatcodeforRNAsthatguideeditingof
themaxicircleRNAtranscripts(7).ThekDNAminicircles
of the compounds are sequence dependent, but
have a number of unusual properties and structural
generally the topological effects on AAAAA and AA
features, such as short, curved A-tract sequences that are
ATT are similar as are the effects on TTTAA
in phase with the helix repeat so that they always appear
and ATATA. A total of 13 compounds with a var-
on the same side of the double helix and cause global
iety of structural differences were evaluated for
bending of the helix (7–9). Given these unusual structural
topological changes to DNA. All compounds features, it seems likely that topological changes in the
decrease the mobility of the ATATA sequence DNA minicircles induced by minor groove binding com-
that is consistent with decreased minor groove poundscanleadtokinetoplastdestructionduringopening
width and bending of the relatively straight DNA of minicircles for transcription and replication and, thus,
into the minor groove. Similar, but generally play a part in the antiparasitic activity of the compounds
smaller, effects are seen with TTTAA. The intrinsic- (1). Binding-induced changes in DNA topology are also
important in the regulation of transcription of many
ally bent AAAAA and AAATT sequences, which
genes, and modulation of bending can enhance or
have more narrow minor grooves, have smaller
reduce transcription (10–12). Small molecules that can
mobility changes on binding that are consistent
affect DNA bending in different ways at selected sites
with increased or decreased bending depending
could, thus, provide tunable controls on the expression
on compound structure.
of the target genes.
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
Present addresses:
Mohamed A. Ismail, Department of Chemistry, College of Science, King Faisal University, P.O. Box. 380, Hofuf 31982, Saudi Arabia.
Reem K. Arafa, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, 11562 Cairo, Egypt.
(cid:2)TheAuthor(s)2011.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
by-nc/2.5),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
4266 NucleicAcidsResearch,2011,Vol.39,No.10
In order to test the hypothesis that compound- opposite that of the DNA during electrophoresis.
induced topological changes in kDNA are important for Including the compound in the gel (Method 1), however,
their antiparasitic activity, a polyacrylamide gel electro- limits the experiment to analysis of one compound at one
phoresis (PAGE) ligation ladder assay (13–17), with concentration per gel.
phased AAAAA bent sequence and ATATA relatively The interesting DNA topological changes observed on
unbent sequences, was established to determine whether binding of DB75 and analogs suggested that it would be
heterocyclic compounds can cause topological changes worthwhile to evaluate additional compounds as well as
in DNA (18). Out of phase ligation ladders of the same DNA sequences to determine the generality of the obser-
ATsequenceswereusedascontrols.Ligationladderswith vations. Such studies will also help to establish relation-
repeated identical binding sites in phase with the helix ships for compound structural effects on the topology of
twistamplifyandsimplifytheanalysisofcompoundtopo- kDNA and other biologically important sequences. In
logical effects at a specific sequence and different binding order to increase the number of compounds and DNA
sites can be evaluated in the same context. The results sequences that can be evaluated per gel, a method that
in the initial assay showed quite clearly that the A-tract does not use compound in the gel is needed. Here a dif-
sequences were slightly straightened by DB75, while the ferent PAGE method (Method 2), without compound in
compound significantly bent the alternating AT DNA the gel, has been tested. Method 2 was tested with com-
pounds of varied structure (Figure 1), and binding con-
sequence (18). Compounds with single atom changes in
stants were determined to establish the limits for
DB75 were also evaluated by replacing the furan with
application of the method. The PAGE method without
thiophene (DB351) and selenophene (DB1213) groups
compound in the gel is able to qualitatively reproduce
and compound-specific effects were observed. In these
the more quantitative results with Method 1, and with
experiments the gel electrophoresis was done with the
Method 2 it is possible to study several compounds at
compound included in the gel at a concentration to
different ratios to DNA sites with different DNAs on
maintainsaturationofbindingsitesduringtheexperiment
one gel.
(Method 1). The method of putting compound in the
gel to help prevent dissociation has been successfully
Compound selection
used with other compounds (19,20). The heterocyclic
diamidines investigated here are positively charged at the Compounds were selected for testing in the screening
typical buffer pH of 8.3, and thus migrate in a direction assay based on systematic modifications to each of the
Figure 1. Compounds grouped by structural similarity.
NucleicAcidsResearch,2011,Vol.39,No.10 4267
molecular units of DB75 (Figure 1). The compounds are DNA oligonucleotides
grouped based on common structural motifs (Figure 1).
Two21-merduplexes(AAAAAandATATAinFigure2)
The central furan of DB75 was replaced with a pyrrole
fromTevisetal.(18)andtwonewduplexes(TTTAAand
(DB262) and from that structure the terminal amidines
AAATTinFigure2)wereusedinthisstudy.Themarker,
were changed to reversed amidines (DB884). One phenyl
M , is also a 21 mer with the same base composition
was changed to a benzimidazole (DB293) and from that 1
as the test duplexes, however; there is no AT binding
compound the furan was changed to an imidazole
site (18). Marker, M , is a commercially available 20bp
(DB940), or the other flanking phenyl was replaced with 2
ladder(BayouBiolabs,Harahan,LA,USA)withadouble
a furan (DB1003). Another group had one amidine
intensity band at 100bp. High-performance liquid chro-
replaced with an amide (DB1932 and DB1933) such that
matography(HPLC)purifiedandlyophilizedDNAoligo-
theyweremonocations,notdications.DB185isadifferent
merswerepurchasedfromIntegratedDNATechnologies,
structure that is related to the well-known bisbenzimi-
Inc. (IDT, Coraville, IA, USA). The concentration of
dazole, Hoechst 33258 (Figure 1). A final group of
single-strand DNA was brought to (cid:3)1mM using water.
compounds has one phenyl of DB75 changed to
The exact concentration was then determined using the
benzimidazole and the furan converted to a phenyl. In
extinction coefficient calculated by the nearest neighbor
this group DB911 has the substituents in a meta position
approximation. Then the complementary strands
at the central phenyl to give a classically curved minor
(100mM) were combined in a 1:1 ratio based on the
groove binding shape, while an isomer has the central
determined concentrations and annealed in 1(cid:4) ligation
phenyl para substituted (DB921) to give a relatively
buffer (New England BioLabs, Ipswich, MA, USA) con-
linear compound. Two analogs of DB921, one with the
taining 50mM Tris–HCl, 10mM MgCl , 10mM
addition of a hydroxyl group (DB985) and one with the 2
dithiothreitol, 1mM ATP and 25mg/ml bovine serum
position of the terminal phenyl amidine group in a meta
albumin.
position (DB1055), are included in this group.
Ligation ladders
MATERIALS AND METHODS The ligation ladder protocol is the same as reported by
Maher et al. (15,27,28) and Tevis et al. (18). Annealed
DNA binding compounds
duplexes (100mM) were 50-phosphorylated using T4 poly-
Compounds in Figure 1 were synthesized by previously nucleotidekinase(NewEnglandBiolabs)andthenligated
described methods (21–26), while the synthesis of usingT4DNAligase(NewEnglandBiolabs)inthebuffer
DB940, DB1003, DB1932 and DB1933 will be published provided. The kinase reactions were 200ml of 6mM DNA
elsewhere. All the compounds were prepared in a 1-mM with 80U of T4 polynucleotide kinase. The reaction
stock solution and stored at 4(cid:2)C away from light. was incubated for 30min at 37(cid:2)C followed by enzyme
Figure 2. Sequence of DNA duplexes used (left) and 8% PAGE gel of DB75 with the duplexes (right). The colored arrows indicate the 189-bp
duplex for each ladder. The red arrow of M indicates 100bp, while the red arrow of M indicates 189bp of the random sequence marker.
2 1
Electrophoresis was done for 2h 20min at 200V and compound was not included in the gel. For each duplex, 0 indicates DNA that is not
incubated with compound prior to electrophoresis, and the ratio 2:1 (compound to binding site) indicates DNA incubated with compound prior
to electrophoresis. The brightest bands near the top of the lanes are circular products.
4268 NucleicAcidsResearch,2011,Vol.39,No.10
deactivation for 30min at 65(cid:2)C. Ligation reactions were Calculating R
L
200ml using 1200 U of ligase and ligation was done at
The analysis of topology changes was done with
room temperature for 20min followed by enzyme deacti-
ImageQuantTL (Amersham Biosciences, Piscataway, NJ,
vation for 25min at 65(cid:2)C.
USA).Fromthebasepair(bp)assignmentstheparameter
R , where R =L /L , was calculated
L L apparent actual
Gel electrophoresis (18,27,28). The mobility of each band of the standard,
M , was determined and correlated with its length, in bp
Ligation ladders were separated using 8% native poly- 1
(L ) and molecular weight. The mobilities of each band
acrylamide (1.5mm thick, 20cm long) prepared from a act
of the test DNAs and their complexes with diamidines
40% acrylamide stock solution (29:1 bis-acrylamide:-
were next determined and correlated to apparent bp
acyrlamide; EMD, Gibbstwon, NJ, USA) in 1(cid:4) TBE
lengths (L ) and molecular weights based on the
buffer (0.089M Tris, 0.089M boric acid, 2.0mM EDTA, apparent
standard values. The R value for each unbound DNA
pH 8.3). Each sample had a final volume of 20ml, which L
duplex was normalized to 1.00 so that the changes
contained the following: 10ml of 2mM DNA, 4ml of
induced by compound binding could be more easily
6(cid:4) load dye (Promega, Madison, WI, USA), the volume
compared within each sequence.
of compound needed for the desired ratio of compound
to binding site and double distilled water to bring to the
Biosensor-surface plasmon resonance studies
final volume, if needed. For the 1:1 and 2:1 ratios 3 and
6ml of compound (13.3mM) were added, respectively. AAAAA, ATATAT, AAATT and TTTAA hairpin
Electrophoresis was done at 200V (10V/cm) for 140min duplexes with 50-biotin groups were purchased from IDT
at 25(cid:2)C±0.1(cid:2)C in 1(cid:4) TBE buffer. The quite different (Integrated DNA Technology) with HPLC purification
observed mobilities with compounds in adjacent lanes and mass spectrometry characterization. The oligomer
indicate that when using the 140-min time period, there sequences are all as follows: GCGWWWWWCGTCTC
is no significant interference from inter-lane diffusion CGWWWWWCGC, where the W sequences are AAAA
when different compounds are used in the gel lanes. The A(cid:5)TTTTT, ATATAT(cid:5)ATATAT, AAATT(cid:5)AATTT, TTT
lack of interference is also supported by the observation AA(cid:5)TTAAA and the hairpin loop is underlined. A 1-mM
that compounds have the same mobilities when studied stock solution of each compound was prepared in water
alone(Figures2and3)oronagelwithothercompounds and diluted to working concentrations with buffer imme-
(Figure 4). diately before use. Surface plasmon resonance (SPR)
After electrophoresis, gels were stained with SYBR measurements were performed with a four-channel
Gold Nucleic Acid stain (Invitrogen, Carlsbad, CA, Biacore 2000 optical biosensor system by using published
USA) at the concentration and time recommended by procedures (29,30). For the duplex binding deter-
the manufacturer. The gels were imaged using an minations, the 50-biotin-labeled DNA samples were
UltraLum Omega 10 gD Molecular Imaging System immobilized in separate flow cells on streptavidin-coated
(UltraLum, Claremont, CA, USA). sensorchips(BiacoreSA)withoneflowcellleftblankasa
Figure 3. (A)PAGE(8%)ofcis-ligatedsequenceswithDB921notinthegel.ElectrophoresiswasdoneasinFigure2.Foreachduplex,0indicates
DNA that is not incubated with compound prior to electrophoresis, and the ratios (compound to binding site) indicate DNA is incubated with
compoundpriortoelectrophoresis.Thecoloredarrowsindicate189bpforeachduplex.TheredarrowofM indicates100bp,whiletheredarrowof
2
M indicates 105bp of the random sequence marker. (B) PAGE (8%) was done for 2h 20min at 200V and compound was included in the gel.
1
Experiments were done with the same cis sequences as in (A) and with trans-ligated DNA sequences of AAAAA (tA ) and ATATA (tAT). The
5
colored arrows indicate 189bp and the double intensity band of M is 100bp.
2
NucleicAcidsResearch,2011,Vol.39,No.10 4269
Figure 4. PAGE (8%) of the DNA sequences (Figure 2)with multiple DB compounds. (A) The duplexes AAAAA and ATATA with five selected
compounds, and(B) TTTAAandAAATT withthesamefivecompounds. Foreachduplex,DNAs areincubatedwithcompound priortoelectro-
phoresisataratioof2:1(compoundtobindingsite).Thecoloredarrowsindicate189bpforeachduplex.TheredarrowM indicates105bpofthe
1
random sequence marker. Electrophoresis was done as in Figure 2 and compound was not included in the gel.
control. The SPR experiments were performed at 25(cid:2)C in DNAs without DB75, and the results are very similar to
filtered, degassed 10mM cacodylic acid buffer (pH 6.25) those previously reported for the same sequences with
containing 100mM NaCl, 1mM EDTA and 0.005% sur- the compound in the gel (18). Two new duplexes, TTTAA
factant P20. Compound solutions were prepared by serial and AAATT, are also included in Figure 2 and the
dilution from the stock solution and injected from 7-mm pink arrows show that DB75 does not have a detectable
plastic vials with pierceable plastic crimp caps at the flow topological effect on TTTAA under these conditions,
rate of 25ml/min. The compound was then replaced by while the orange arrows show that DB75 increases the
buffer flow to monitor dissociation of the complex. The mobility of AAATT to an even greater extent than with
reference response from the blank cell was subtracted A5. There are two alternative explanations for the
from the response in each cell containing DNA to give a mobility changes observed for DB75 and the other com-
signal(responseunits,RU)thatisdirectlyproportionalto pounds in this study; the compounds could be changing
theamountofboundcompound.Sensorgrams,RUversus the bend angles of the DNAs or they may be slightly
time, at different concentrations for binding of each changing the helical repeat, resulting in improved or
compound to DNA were obtained, and the RU in the decreased phasing of the AT repeats. Because the trans
steady-state region was determined by linear averaging A5 sequence is already significantly dephased, an
over a selected time span. The number of binding sites
increase in the mobility of the A5 ladder is probably due
and the equilibrium constant were obtained from fitting
to a decrease in the curvature of the A5 A-tracts. In the
plots of RU versus C . Binding results from the SPR
free same manner the dramatic reduction in mobility of the
experiments were fit with either a single site model
ATATA sequence is highly unlikely to result from a
(K =0) or with a two-site model:
2 phasing change because the sequence is straight no
r¼ðK (cid:4)C +2(cid:4)K (cid:4)K (cid:4)C2 Þ= matter whether it is phased or not (18 and cis-trans in
1 free 1 2 free ð1Þ Figure 3). It seems likely then that bending rather than
ð1+K1(cid:4)Cfree+2(cid:4)K1(cid:4)K2(cid:4)C2freeÞ dephasingofhelicalrepeatsisresponsiblefortheobserved
changes in mobility for these DNAs and compounds.
whererrepresentsthemolesofboundcompoundpermole
Additional experiments with 20- and 22-bp duplexes
of DNA hairpin duplex, K and K are macroscopic
1 2 would be required in order to definitively differentiate
binding constants and C is the free compound concen-
free between phasing and bending or straightening of the
tration in equilibrium with the complex.
AT sequences to account for the mobility changes.
The brightest bands near the top of each lane are
RESULTS circularproductsthatappearforallDNAsasthemolecu-
lar weight increases. The circular bands appear at lower
Topological screening methods: comparison of two assays
molecularweightsforthebentDNAs,AAAAAandAAA
In Figure 2 the A5 sequences show increased mobility TT than for the more linear duplexes. As can be seen in
(green arrows), while the ATATA sequences have a the figure, the compounds have very little effect on the
reduced mobility (blue arrows) relative to the same mobilities of the circular DNAs. As compounds are
4270 NucleicAcidsResearch,2011,Vol.39,No.10
added, the linear bands can change markedly in mobility Using R to compare topological changes
L
while there are no detectable changes for the circular
R values for each DNA and complex were calculated
DNAs. The potential significance of this for biological L
relative to the mobility of the standard as described in
activity is presented in the ‘Discussion’ section.
the ‘Materials and Methods’ section. Table 1 compares
Figure 3 shows an additional comparison of gels done
R values of several compounds that were determined in
with DB921 by Method 1, which has DB921 in the gel L
bothassays.ThetopologicalchangesshowninTable1are
(Figure 3B), and the new Method 2, where DB921 is
similarbybothassays,however;forsomecompoundsand
added only to the separate DNAs and is not in the gel
sequences the effects on R are larger by Method 1. This
(Figure 3A). DB921 has a very different structure from L
difference is not unexpected from the results in Figure 3
classical minor groove binders with a more linear shape
and as noted above, is due to partial dissociation of
that incorporates an interfacial water molecule to bind in
the complex in the screening assay (see binding results
theminorgroove(31).Ascanbeseen,DB921reducedthe
below). The trend and general magnitude of the changes,
mobility of all four of the cis-ligated duplexes of interest
however,canbedeterminedwithoutcompoundinthegel.
bysimilaramountsinbothassaysandtheresultsarequite
For quantitative determination of bending angle changes
different from those with DB75 (Figure 1; 18). This
on compound binding, however, Method 1 should be
example shows that both methods give equivalent results
used.
for compounds of quite different structure. Interestingly,
To assist in comparing results for different DNAs, all
for the TTTAA and ATATA sequences there is a
R values were normalized by dividing them by the R
smearing of the bands in Figure 3A that is not present L L
value of the free DNA, and the results are plotted in
in Figure 3B and this smearing appears to be the result
of partial dissociation of the DNA-compound complex Figure 5. A compound with an RL value greater than 1
during the gel experiment. In order to help clarify this indicates the sequence had lower mobility than the free
difference, binding constants (see below) were determined DNAs and is consistent with bending, while a value
forDB921andshowthatitbindsmoreweaklytoTTTAA below one is consistent with straightening of the
andATATAthantoAAAAAandAAATT.Theseresults sequence. Interestingly, the ATATA sequence was bent
explain the smearing of DB921 bands in Figure 3A by all compounds tested although the magnitude of
because it will more easily dissociate from the TTTAA bending varied with compound structures. DB921 and
and ATATA DNAs during the gel experiment. Note,
however, that the general effects of DB921 on all the
DNAs are comparable in both methods and can be Table 1. R values for both assays for each DNA sequence
L
determined from the screening method.
AAAAA TTTAA AAATT ATATA
Forcomparisonwithourpreviousresults(18),Figure3
also includes the trans AAAAA and ATATA sequences.
Drug C No C C No C C No C C No C
WithDB75weobservedmuchsmallereffectsonboththe
bent AAAAA sequence and the straight ATATA DB75 0.77 0.82 1.20 1.01 0.75 0.86 1.31 1.19
sequence. DB921 reduces the mobility of the trans DB262 0.92 0.95 1.25 1.06 0.76 0.81 1.48 1.27
DB884 1.22 1.11 1.16 1.08 1.05 1.03 1.19 1.13
duplexes, but the effects are much less than with the cis
DB921 1.1 1.12 2.1 1.33 1.09 1.08 2.5 1.9
sequences, as expected.
Cindicatescompoundinthegelmatrix(Method1),andNoCindicates
Effects of compound structure on DNA topology nocompoundinthegelmatrix(Method2).TheMethod2comparison
usestheR valuesatthe2:1ratioofcompoundtobindingsite.
L
In Figure 4, PAGE results for two DNAs and multiple
compounds at a fixed ratio are compared. Topological
effects of the compounds used in these gels agree with
the results in Figures 2 and 3 and Supplementary Data.
The screening method with multiple compounds, thus,
allows for rapid testing and direct comparisons of topo-
logicalchangesforcompoundsinalibrary.InFigure4the
2:1 ratio is used, because smaller effects are seen at a 1:1
ratioandgoingtoa3:1ratiodidnotgenerallycauselarge
changes in mobility with these DNA sequences and com-
pounds. The PAGE results for all the other compounds
inFigure1canbeseeninSupplementaryData.Inthegels
forDB1003andDB185(SupplementaryFiguresSM4and
SM10, respectively) additional ratios were included and
none of the extra ratios changed the conclusions. To
accommodate the extra ratios, the ATATA sequence
was omitted for DB1003 and DB185. These two com- Figure 5. Histogram showing RL values for all compounds with
AAAAA, ATATA, TTTAA and AAATT sequences. The blue repre-
pounds were selected for this test because DB1003 is a
sents AAAAA, red ATATA, the green TTTAA and the purple
relatively weak binder (32), while DB185 is a very strong
AAATT. Each group represents a structural group from Figure 1.
minor groove binding agent (33). All the data shown are for a 2:1 ratio of compound to binding site.
NucleicAcidsResearch,2011,Vol.39,No.10 4271
DB911 show the largest induced bending, followed by
DB262 and DB75. All three of the monocations caused
asmallreductionofmobilityandasmallbendwiththeA
TATA sequence.
ThechangesinR fortheAAAAAsequencewereboth
L
positive and negative and were generally smaller than
the changes with ATATA. The following compounds
cause a decrease in mobility, consistent with increased
bending of the AAAAA sequence: DB1933, DB921,
DB1003, DB911 and DB884. DB75 and DB185, on the
other hand, show an increase in mobility and DB262, the
pyrrole analog of DB75 increase mobility but to a lesser
extent. Interestingly, the monocation, DB1933, causes the
largest decrease in mobility in AAAAA. DB884, with a
reverse amidine cation, also decreased the mobility of the
intrinsically bent AAAAA sequence. The compounds
DB293, DB940, DB985, DB1055 and DB1932 did not
have a significant topological effect on the AAAAA
sequence. In general, the results with AAATT are
similar to those with AAAAA.
For the TTTAA sequence the group containing the
phenyl-benzimidazole modification (Figure 1) shows the
largest effect on bending. DB921 and DB911 increase
bending more than DB1055 that bends more than
DB985. DB262 increased the degree of bending more
than DB75. For the benzimidazole modification group,
DB293 caused the largest bending effect with TTTAA
and the bis-benzimidazole DB185 shows a similar effect.
The effects with DB75, DB940, DB1003 and DB1932 are
not significant for the sequence.
Biosensor-SPR determination of equilibrium binding
constants (K)
Binding assays were conducted for the compounds from
thetopologicalstudiestoanswertwoimportantquestions:
(i) what is the minimum K value that is necessary before
Method 2 can be used without compound in the gel and
(ii) is there any correlation of the K and R values.
L
Because three biotin-labeled DNAs can be immobilized
on four-channel Biacore streptavidin-coated sensor chips
withoneflowcellleftasablank(29,30),twosensorchips
were used to evaluate K values for the four DNA
AT-binding sequences used in the topology-ligation
assays. This provided repeat values for two DNAs to Figure 6. Examplebiosensor-SPRsensorgrams(instrumentresponsein
RU values versus time) for binding of DB262 to (A) ATATA and
give a better evaluation of reproducibility of K values
(B) AAATT at 25(cid:2)C. The unbound compound concentrations in the
determined with completely different experimental sensor
flowsolutions rangefrom0.005mM inthelowest curve to1mM inthe
chips, samples, etc. The variation in K is <10% in the top curve. Experiments were conducted in cacodylic acid buffer with
repeat assays. 0.1M NaCl. (C) SPR binding plots for DB262 in the presence of
Example sensorgrams for DB262, which has similar ATATA (top), AAATT (middle) and TTTAA (lower). The RU
values from the steady-state region of SPR sensorgrams are plotted
structure and DNA binding to DB75, show that the
versus the unbound compound concentration. The RUs and the con-
compound has relatively fast association and dissociation centrations of DB262 are from repeat experiments. The lines in the
kinetics for binding to ATATA and AAATT (Figure 6A Figure are for one or two-site fitting (‘Methods’ section). K values
and B). In this case K values can be determined by fitting for AAATT were obtained by using a one-site model and for
ATATA and TTTAA with a two-site model to get K and K . K
the kinetic constants for association (k ) and dissociation 1 2 2
a wasmuchlowerthanK andonlyK valueswereusedinthecompari-
(k ) or from the steady-state response where the rates of 1 1
d son plot in Figure 7.
association and dissociation are equal (Figure 6A and B).
The steady-state method is not subject to any mass trans- in Figure 6C to one or two site functions as described in
port issues at the sensor surface, and steady-state RU the ‘Materials and Methods’ section. In all cases the K
1
values for DB262 and three DNA sequences are plotted values are significantly larger than the K values when a
2
inFigure6C.Kvaluesweredeterminedbyfittingtheplots two-site fit was required, for example, with the ATATA
4272 NucleicAcidsResearch,2011,Vol.39,No.10
and TTTAA DNAs (Figure 6C). A simple two-site constants with the mobility changes for the compounds
binding function can be used in these cases because the and DNAs.
primarybindingisforcomplexformationattheATtarget
sequences while the much weaker, secondary binding is
generally a non-specific electrostatic interaction (34). DISCUSSION
Results for DB921, which has slower association and dis-
Experiments with ligation ladders from DNA sequences
sociationrates,areshowninSupplementaryFigureSM15,
withAAAAAandATATAbindingsiteshaveshownthat
Supplementary Data. K values for DB921 and related
DB75 and some close analogs induce topological changes
compounds were determined by global fitting of the
inDNAthataresensitivetoDNAsequence/structureand
sensorgrams to determine k and k values as a function
a d to compound structure and properties (18). At present
of the compound concentration in the flow solution.
there is considerable knowledge of the effects of protein
DB921, DB985 and DB1055 are generally quite strong
binding on DNA topology but much less information
minor groove binding compounds. For visualization,
aboutsmallmoleculesthattargetDNA.Suchcompounds
primary binding constants (K ) are compared for repre-
1 are being designed for therapeutic applications that
sentative compounds as a histogram in Figure 7, and the
range from antiparasitic to anticancer activities (1–4),
actual K values are tabulated in Supplementary Figure
and in order to develop these agents in a rational way
SM15.
we need to understand the topological effects at the
As can be seen, there is a large variation in binding
target sequences. Topological changes on binding of the
constants for the compounds with different DNAs. In heterocyclic diamidines of Figure 1 to the mitochondrial
general, the highest binding constants were observed kinetoplast DNA of disease-causing eukaryotic parasites
with the AAATT site and the lowest with the reversed maybeakeystepindestructionofthekinetoplastandthe
sequence TTTAA. Significant outliers in this pattern are biological mechanism of action of the compounds (3). To
DB1003 and DB1932, which bind best to TTTAA as clarify this question, more topological information on the
expected for compounds with that structure (32). The effects of the compounds on kinetoplast target sequences
compounds with the largest K values in this group are is required.
DB921 and analogues, but they also have relatively low Method 1, with compound in the gel to prevent the
K values for TTTAA and the highest values for AAAAA cationic compounds from dissociating from DNA during
and AAATT. The two pyrrole compounds, DB262 and electrophoresis,hasbeendevelopedforquantitativeevalu-
DB884, have quite large values for AAATT relative to ation of compound-induced topological changes on
their Ks for the other three DNAs. Most compounds complex formation (18). This method, however, allows
have larger K values for the non-alternating, AAAAA, only one compound at one concentration (ratio per
than for the alternating ATATA sequence. Interestingly, DNA binding sites) on each gel. In this work, Method
the diamidines, DB75 and DB262, with a diphenyl- 2,inwhichthecompoundsareaddedtoDNAatdifferent
5-member ring structure, have larger values for ATATA. ratios but are not in the gel, was tested. This method
AswillbediscussedbelowandasshowninSupplementary allows the possibility of different compounds on each gel
Figure SM16, there is no direct correlation of binding and on a single gel with 20 lanes, for example two ratios
of different compounds can be tested with up to four dif-
ferent DNAs, free DNA and appropriate standards.
Figure3,withonecompound,andTable1withfourcom-
pounds and four DNAs compare results by Methods 1
and 2. In Table 1 the changes observed by Method 1 are
generallygreaterthanthosewithMethod2,asexpectedif
thereissomecompounddissociationandlossduringelec-
trophoresis. Partial dissociation is supported by results in
Figure3,wheresomesmearingisseeningelA(Method2)
withtheTTTAAandATATAsequences,butnosmearing
is observed in gel B (Method 1). The smearing effect
is observed with compounds and DNAs where there is
a large difference in R with a relatively low K value.
L
The smearing, for example, occurs with the TTTAA and
ATATA sequences, and DB884, which has lower binding
constants with these DNAs than with AAAAA and
AAATT (Figure 7 and Supplementary Figure SM1). The
encouragingaspectofthesecomparisons,however,isthat
in all cases the direction of the changes and general
relative magnitude is reported identically by the two
methods. These results indicate that Method 2 can be
used for qualitative analysis of induced topological
Figure 7. Histogramsofequilibriumconstants,K(M(cid:6)1)forrepresentative changes by small cations that target DNA. For com-
compoundsbinding to ATATA, AAAAA, AAATT andTTTAA. pounds that are found to be particularly interesting from
NucleicAcidsResearch,2011,Vol.39,No.10 4273
theseresults,morequantitative,butslower,studiescanbe ATATA is that all minor groove binders studied to date
done by Method 1 as necessary. cause bending of that sequence. The magnitude of
With these encouraging results for Method 2, several bending, however, depends on the interaction of the
groups of minor groove binding compounds of quite dif- compound structure and chemical groups with the ATA
ferent structures and properties (Figure 1) were selected TAgroove.TheTTTAAsequencealsohasawidergroove
for a more extensive, comparative analysis of induced thantheA-tractsequencesandithasamuchlowerdegree
topologicaleffectsoncomplexformation.Thesetofcom- of bending (41,42). Compounds also generally show
pounds and DNAs is satisfactory to determine if the bending on complex formation with this DNA, but the
induced changes are dominated by either compound bending magnitude is lower than with ATATA. This
or DNA structure and properties, and also to look for could be due to the fact that compounds, such as
any patterns in the results. A qualitative conclusion DB1003 and DB293, have been shown to bind to the
from the results that are summarized in Figure 5 for R TTAA sequence as a dimer (32), which will maintain a
L
and Figure 7 for K is that there is no direct correlation wider groove.
betweenthemagnitudeanddirectionoftheinducedtopo- It is a very interesting result that the circular DNA
logical changes and the binding affinity, as shown in bands in Figures 2–4 (the bright bands in the upper part
Supplementary Figure SM16. As will be described of the gels, see Figure 2) do not change mobility by any
below, the observed topological changes appear to be significant amount when compound is added. This is true
best correlated with DNA intrinsic minor groove width even though they have the same sequence as the linear
as well as with compound shape and properties. DNAs and even when the mobilities of the linear bands
The AAAAA minor groove is intrinsically narrow with change by a large amount. Clearly constraining the ends
ordered water and cations stabilizing the close approach of these sequences is the dominant feature of their shape
of phosphate anions across the groove (35,36) and as a and mobility in PAGE. Because the kinetoplast DNAs
result,thissequenceisthemostintrinsicallybentinourset arecircular,thisobservationsuggeststhatthecompounds
of four DNAs (27,28). The groove is of the correct width would have relatively little effect on structure until the
to bind most compounds of Figure 1 with little change DNAs were opened for transcription or replication by
in width. The compounds can cause a slight increase in mitochondrial enzymes. At this point their topology
bending by stabilizing a slightly more narrow minor could change significantly and closing the DNAs to
groove with increased bending into that groove. reform the kinetoplasts would be inhibited. Given the
Decreased bending, which can be caused by the opposite thousands of minicircles in each kinetoplast (7), even a
effect of a slight widening of the groove, might be gener- small amount of inhibition could lead to very significant
allyexpected,andforthiscompoundsetitisobservedfor destruction of the kinetoplast structure and network.
most complexes. Effects on the AAATT sequence, which In summary, the major features of both induced topo-
alsohasA-tractpropertiesandsignificantbendingforthe logical changes and of compound binding constants for
freeDNA,aregenerallysimilartothoseforAAAAAand minor groove complex formation are dominated by the
can be either in the direction of increased or decreased groove characteristics of DNA AT sequences. There are,
bending. however, major variations within a DNA sequence for
The ATATA sequence is the least intrinsically bent changes in compound structure and properties. The
DNA in our set of four (18,37–42) and this DNA has a screening method described in this report can now be
widergroovethanAAAAAorAAATT.Thegrooveistoo used with a quite large set of DNA binding agents to de-
widetobindtightlytothecompoundsofFigure1without terminecorrelationswithmolecularstructureandinduced
some structural changes. Minor groove binding that de- topological changes.
creases the groove to better fit the general width of the
compoundsshouldbendtheDNAintotheminorgroove.
All compounds of Figure 1 whether mono or dications, SUPPLEMENTARY DATA
withorwithoutabenzimidazolegroup,andwithamidines
Supplementary Data are available at NAR Online.
or a reversed structure (DB884) bend ATATA indicating
that topological effects with this sequence, are dominated
by the DNA structure. The benzimidazole-biphenyl FUNDING
isomers, DB921 and DB911, cause the most bending,
National Institutes of Health (NIAID AI64200 to
and the related compounds, DB985 and DB1055, cause
W.D.W. and D.W.B.). Funding for open access charge:
a smaller but significant amount of bending. The
National Institutes of Health (NIAID AI64200).
biphenyl motif of these compounds is intrinsically
twisted by 30–40(cid:2) and this may explain the relatively
Conflict of interest statement. None declared.
large bending effects of the compound family. Replacing
the central phenyl by a furan, DB293, or with an imid-
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