BACKGROUND
dna microarray technology relies on the highly specific binding affinity of surface-tethered dna probe sequences to complementary target sequences.
nucleic acid hybridization, the sequential base pairing between complementary probe and target strands, results in the formation of stable double-stranded duplexes.
in microarray hybridization assays single-stranded nucleic acid targets – contained in a complex mixture of diffierent target sequences in solution – freely diffuse over the surface-tethered probes until they are captured by a complementary probe.
target strands often carry fluorescent dye labels to enable quantitative detection of the individual target species.
hybridized targets can be identified by the position of the corresponding microarray features  within the regular grid of the dna microarray.
in dna microarray applications, along with a high binding affinity , a high specificity of probe-target hybridization is required to discriminate between sometimes very similar homologous sequences.
binding specificity is particularly important in genotyping applications where single nucleotide polymorphisms , genetic variations of single bases, are concerned.
snps determine genetic individuality, but also predisposition to a variety of genetic diseases, response to drugs, pathogens, chemicals and other agents.
snps are of great interest not only for genetic research but also for medical diagnostics and therapy  <cit> .
snps and point-mutations can be detected by means of relatively short  <dig> to  <dig> mer probes: already a single mismatched  base pair can result in a significant decrease of the duplex binding affinity with respect to the corresponding perfect matching  duplex  <cit> .
the binding affinity of mismatched duplexes – in bulk solution – is commonly predicted on the basis of the nearest-neighbor model  <cit> .
a recent study by pozhitkov et al.
<cit>  revealed a poor correlation between predicted duplex binding affinities and actual hybridization signal intensities implying that the thermodynamic properties of oligonucleotide hybridization on dna microarrays are by far not understood.
in dna microarray experiments the binding affinity of mismatched oligonucleotide duplexes is governed not just by nearest-neighbor parameters – as in solution-phase hybridization – but mainly by the position of the defect  <cit> .
furthermore, the secondary structure of the long target strands  <cit>  and various surface effects  <cit>  have a significant influence on the microarray binding affinity.
our study is a comprehensive approach to understand how point defects affect the hybridization of fluorescently labeled oligonucleotide targets to surface-bound oligonucleotide probes.
rather than previous work on single base mms, which has been conducted with complex target mixtures either from pcr products  <cit>  or in vitro transcripts  <cit> , we employ short , end-labeled oligonucleotide targets, thus avoiding labeling and steric hindrance related effects.
in order to avoid competitive binding  <cit>  we perform each hybridization assay with a single target sequence.
oligonucleotide target sequences  were chosen to minimize secondary structures and any related influence on the hybridization signal.
in particular we investigated differences between the impact of defects on dna/dna and analogue rna/dna duplexes.
fluorescently labeled dna and rna target oligonucleotides.
dna chips were fabricated by light-directed in situ synthesis  <cit>  with a digital micromirror device  based maskless synthesis apparatus  <cit>  developed in our laboratory  <cit> .
sets of probe sequences were derived from probe sequence motifs by systematic variation of defect type and defect position including all single base mismatches, insertions and deletions.
the design of the hybridization experiments  enables discrimination between the strong influence of defect position  <cit>  and the more subtle defect-type and sequence related factors.
after the current article was submitted, we became aware of further related studies in this area.
suzuki et al.
<cit>  performed hybridization on custom nimbleexpress™ arrays  to investigate the influence of the probe length and mismatch position on single base mm discrimination.
fish et al.
<cit>  performed a direct comparison between hybridization signals  from spotted microarrays and measured thermodynamic melting parameters .
they report a linear relation between the duplex free energy and the microarray hybridization intensity.
the focus of the present paper is on the impact of various defect types  on the hybridization signal.
RESULTS
our microarray hybridization experiments performed in this study provide quantitative information on the binding affinity of individual mismatched duplexes by means of the hybridization signal intensity .
since the absolute hybridization signal intensities of the different sequence motifs employed in this study  are subject to a large variation  we compare the mm hybridization signals with the corresponding pm hybridization signals.
our experiments – experimental details  are explained in the methods section – provide a measure for the mismatch discrimination with respect to the corresponding pm binding affinity, rather than an absolute measure for the mm binding affinity.
the discrimination between the hybridization affinity of point-mutated probes and corresponding perfect matching probes depends on the stability of the particular probe sequence.
in agreement with  <cit>  we observed that the   <dig> mer probes are less discriminative with respect to point defects than the shorter  <dig> mer probes.
discrimination is also reduced for sequence motifs stabilized by a higher cg-content.
mm defect position and hybridization affinity
the "defect profile" plots  show that the dominant parameter determining oligonucleotide probe-target-affinity – on the microarray surface – is the position of the defect.
a moving average evidences a trough-like "mean profile" curve .
a parabolic fit can provide a reasonable approximation for the average position dependence obtained from a large number of different sequence motifs  <cit> .
for  <dig> mer duplexes a single base mismatch in the center typically results in 40% of the pm hybridization signal.
however, for individual sequence motifs we found sequence-dependent deviations from the simple position dependence .
the raw signal intensities and probe/target sequences of the experiment are given in additional file  <dig>
influence of the mismatch type in dna/dna duplexes
in the following we use the notation of the mismatch base pair x·y consisting of the mismatched base x in the probe sequence and the base y in the target sequence.
to investigate how the particular mm-types x·y affect duplex stability we measured probe-target-affinities for  <dig> different sequence motifs.
microarray hybridization experiments with single base mismatch probe sets as well as the extraction of their hybridization signals, which reflect duplex stability, are described in more detail in the methods section.
owing to the limited number of available target oligonucleotides we restricted base substitutions to the probe sequences.
the pm hybridization signals of the different  <dig> mer sequence motifs display a strong variation .
since the relative hybridization signal intensities within the individual probe sets are largely unaffected by this variation, we normalize the "defect profiles" by division with their standard deviation.
the resulting database comprising normalized hybridization signals from about  <dig> different single mm probe sequences, enables categorization of the binding affinities according to the mismatch type.
for statistical analysis of mm type and nearest-neighbor influences the superposed positional influence needs to be eliminated by subtraction of the mean profile.
the resulting position-independent defect profile  consisting of influences of defect type and defect neighborhood only is shown in fig.
2b.
the boxplot representation of this data in fig.
<dig> demonstrates that mm-types affecting c·g base pairs  systematically have lower median hybridization signal values than mm-types affecting a·t base pairs .
we compared the mm-type related hybridization signal deviations δimp from the mean mm profiles  to predicted gibbs free energy differences δΔg37∘=Δg37mm∘−Δg37pm∘ between mm and corresponding pm duplexes.
δΔg37∘ were determined from mismatch nearest-neighbor thermodynamic parameters  <cit> .
our analysis  indicates a decreasing trend of the δimp values with increasing δΔg37∘.
moreover, we observed that single base mismatches with two a·t flanking base pairs tend to provide a better mismatch discrimination than mismatches flanked by two c·g base pairs.
dna/dna single base bulge defects
single base insertions and deletions owing to a surplus unpaired base in one of the two strands result in bulged duplexes.
in our experiments  the bulged base is located on the surface-bound probe strand, whereas in duplexes with single base deletions  the bulge is on the target strand.
we discovered that on average the positional dependence of the insertion and deletion defect profiles  is very similar to the positional dependence of mismatch defect profiles .
within one and the same individual defect profile, single base bulge defects originating from single base insertions or deletions display the same positional dependence as single base mismatch defects , qualitatively as well as quantitatively.
on average single base insertion probes provide increased hybridization signals when compared to mm probes or single base deletions .
besides the significantly increased hybridization signals of group ii insertions , this is due to the reduced number of binding base pairs in the mismatched duplexes .
in single base insertions no binding base pair is substituted, but we see that the influence of the inserted base clearly depends on its neighbor.
the individual curves  show deviations from the  mean profile, hybridization signals can be significantly increased over several consecutive defect positions.
in particular base insertions next to identical bases  result in systematically increased binding affinities – in comparison to insertions of non-identical bases .
group ii bulges located near the center of  <dig> mer probes often show hybridization signals with a similar intensity as the corresponding pm probe .
a statistical analysis with a large dataset  comprising hybridization signal data from  <dig> different 20– <dig> mer probes indicates the general validity of the result.
interestingly, systematically increased hybridization signals  have also been observed for certain group i bulges: for g-insertions next to a t  we frequently find increased binding affinities similar to that of group ii bulges.
we further analyzed the degree of correlation between the binding affinities of probes with different insertion bases x and y : a clear correlation appears between the hybridization signals of probes with t- and g-insertions, and also, though less distinct, between a- and c-insertions.
in contrast, we observed an anti-correlation between g- and a-insertions.
dna/dna versus dna/rna mismatch and bulged hybridization
to investigate if the above results from dna/dna hybridization also apply to hybridization of rna/dna duplexes we performed a direct comparison employing dna targets and corresponding rna targets on the same microarray.
we observed that mm discrimination in rna/dna duplexes is similar to mm discrimination in dna/dna duplexes .
a statistical analysis  reveals, however, that purine-purine mms are  somewhat less stable in rna/dna duplexes than in dna/dna duplexes.
the most significant differences between rna/dna and dna/dna mms  are observed for the mm-types g·a and a·g  and for the mm-type t·g .
a presumed destabilizing effect of purine-purine mms in the ranking order of rna/dna mismatch discrimination  is superposed to the affected base pair effect , which is very similarly, also observed in dna/dna hybridization.
for bulged duplexes we did not observe significant defect-type specific differences between rna/dna and dna/dna hybridization.
the hybridization signal and sequence data from the microarray hybridization experiment are provided in additional file  <dig>
single base insertion, deletion and mismatch defects in comparison
defect profiles for mms and base bulges  exhibit a very similar quantitative influence from defect position in dna/dna as well as in dna/rna complexes.
for individual sequences the mean trough-shaped profile can be altered: fig.
<dig> shows deformations of the trough-like profile on scales much larger than the size of a base pair.
single base mm discrimination also depends on the type of mm base pair and the corresponding pm base pair .
hybridization signals of mms  originating from c·g base pairs are about 25% smaller  than for mms from a·t base pairs.
single base deletions affecting c·g base pairs result in about 30% smaller hybridization signals than deletions affecting a·t base pairs.
the deletion profile in fig.
<dig>  shows that the local ups and downs of the profile curve correlate with deletions affecting either a·t or c·g base pairs.
thus, for mms and single base deletions it is the type of base pair affected by the point-mutation, which determines the impact on hybridization affinity to an important degree, however, it is still less important than defect-position.
we also observe a noticeable influence of the next-neighbor bases of the mismatch .
discussion
dominating influence of defect position
we observe that defects located in the center of the oligonucleotide duplexes are significantly more destabilizing than defects at the ends  <cit> .
similar influence of the mm position has been reported previously from other microarray based studies  <cit> , and also – although sparsely – from hybridization experiments in solution  <cit> .
the limited data in solution may be due to the technical difficulty of studying a large number of different probes.
quantitatively, in accordance with  <cit>  we have identified mm position  as the strongest influential factor on the hybridization signal, when compared to mm-type and nearest neighbor effects.
the well-established two-state nearest-neighbor model, which has proved to be reliable for the prediction of duplex stabilities in solution-phase, does not regard the position of the  nn pairs  <cit> .
we propose that a model for the prediction of microarray binding affinities should also include the position of the nn pairs – in particular in case of mismatched nn pairs.
affinity models for microarray hybridization considering a positional dependence of the nearest-neighbor parameters have been previously discussed in  <cit> .
we observe a very similar position dependence for single base bulge defects as for single base mismatches.
also, the magnitudes of the impacts of the mms and base bulges on the hybridization signal are very similar .
this consistency suggests a common origin of the positional influence, independent of defect type.
sterical crowding at the surface, as suggested by peterson et al.
<cit> , can in principle reduce the accessibility of the probe surface-bound 3'-ends and can thus decrease the impact of defects located near this end.
however, in our case we observe largely symmetrical intensity profiles with respect to both ends of the probes .
focusing on individual probe sequence motifs we observe, that the positional influence does not only depend on the defect-to-end distance, but also has a sequence-dependent contribution.
this indicates that the impact of a defect also depends on the stability of the local sequence environment .
since there are no long range molecular forces, we infer that the molecular dynamics must play a role, effects like breathing bubbles or zipping could be at the origin.
this influence of the duplex sequence and the observed symmetry of the defect positional influence with respect to the duplex ends suggest that end-domain opening  must be suspected to be a key mechanism for understanding the influence of defect position on duplex stability.
influence of the mm-type
removing the positional influence in our data, we see that single-base mms introduced at the site of a c·g base pair result in a larger decrease of the hybridization signal  than mm defects affecting a·t base pairs.
the same applies for single base deletions .
these experimental results , in accordance with nearest-neighbor thermodynamic parameters for watson-crick base pairs  <cit> , mainly reflect the increased base stacking and hydrogen bonding interactions of c·g base pairs.
we observe a positive correlation between the experimentally determined single base mismatch discrimination and predicted free energy increments δΔg37∘  on the basis of the nearest-neighbor model – for details see additional file  <dig>  a similar correlation  hybridization signal values and δΔg37∘) has been reported previously in  <cit> .
we emphasize the good correlation between our dna/dna mm stability order  and the corresponding results of wick et al.
<cit>   hybridization signal values in fig.
5a in  <cit> ).
a major difference, however, occurs for the mm-pair g·g, which is the least stable in our study.
wick  found g·g to be one of the most stable mms.
interestingly, however, pozhitkov et al.
<cit>  – in accordance with our results -identified g·g as one of the least stable mm-types.
our direct comparison between dna/dna and rna/dna hybridization on microarrays reveals – for rna/dna duplexes – an increased destabilization of purine-purine mismatches, with respect to other mm types.
an explanatory approach for the observed differences between dna/dna and rna/dna binding affinities is, that purine-purine mms cause larger steric hindrance in the a-form hybrid duplexes than in the b-form dna/dna duplexes.
in contrast to  <cit>  we did not observe that purine-purine mismatches in rna/dna duplexes are, in absolute terms, more discriminative than other mm-types.
increased stability of group ii single base bulges
we observe significantly increased hybridization signals of single- base insertion defects in which the insertion base is placed next to a like-base.
our investigation shows that  the difference between group i and group ii binding affinities δibulge  is distinctly larger than the defect-type related variation of binding affinities δimm .
for comparison, the free energy differences among the mm trinucleotide duplexes abc/a¯xc¯ and abc/a¯yc¯  span the range δΔgmm <dig> =  <dig>  to  <dig>  kcal/mol .
the increased stability of group ii bulges in comparison with group i bulges has been investigated previously in solution rather than on microarrays  <cit> .
according to ke and wartell  <cit>  the increased stability of group ii bulges originates from positional degeneracy of the base bulge.
additional conformational freedom, entailing higher entropy, results in lowered duplex free energy .
according to zhu et al.
<cit>  position degeneracy accounts for an average stabilization of - <dig>  to - <dig>  kcal/mol  for a two-position degeneracy.
znosko et al.
<cit>  reported group ii duplexes to be on average δΔg <dig> = - <dig>  kcal/mol more stable than group i duplexes.
the latter value matches better our observation of group ii hybridization close to the perfect match hybridization signal.
for explanation of the large binding affinity of group ii duplexes we propose the following mechanism  based on the molecular zipper model  <cit> : even in thermal equilibrium due to thermal excitation, zipping  as well as unzipping occur.
the extend of the end-domain denaturation of the duplex, which is described by a random walk , may finally result in complete dissociation of the duplex.
the binding affinity is determined by the ratio knuc/kdiss between the nucleation rate knuc and the duplex dissociation rate kdiss.
we consider that a defect does not have an important influence on the unzipping, since the defect does not present a barrier for the process.
for closing of the strands, however, the situation is different.
the surplus  base must act as a kinetic barrier, interrupting the rapid zipping of the duplex.
the 1-nt frameshift between the  complementary strands, owing to the unpaired bulge base prevents closing beyond the defect and results in a partially zipped, and correspondingly weakly-bound, duplex.
duplex closure can only progress if the interfering surplus base is giving way , thus allowing the subsequent base to form a watson-crick base pair with the complementary base in the target strand.
from this point zipping can progress rapidly.
therefore compared to watson-crick nearest-neighbor pairs, a base bulge  has a decreased ratio of zipping/unzipping-rates k+/k- and thus favors unzipping of the duplex .
for group ii bulges the k+/k- ratio at the defect site is increased with respect to group i bulges: in case of a group ii bulge there is an increased probability that any of the degenerate bases makes way  while simultaneously the subsequent base forms a base pair.
this is due to the increased number of possible molecular conformations, which can lead to continuation of the zipping.
then, as the frameshift is compensated, the rapid zipping to complete the duplex occurs.
since the nucleation rate knuc of group i and group ii duplexes may be assumed to be the same, the binding affinity of group ii duplexes must be increased.
previous studies – including rna/dna hybridization
tautz and coworkers  <cit>  performed a mismatch study with  <dig> mer oligonucleotide microarrays fabricated by light-directed in situ synthesis with the geniom® one instrument .
similar as in our study, they compared normalized hybridization signal intensities.
however, an important difference between the experiments described in  <cit>  and our experiments is the use of in vitro transcribed rna targets  <cit>  originating from ribosomal rna.
they observe a more pronounced destabilization by purine-purine mms compared to our results.
a further study on the impact of mm stabilities in rna/dna duplexes, in solution rather than on a microarray surface, has been published by sugimoto et al.
<cit> .
as discussed in  <cit>  the destabilizing effect of purine-purine mms is not observed by sugimoto et al.
<cit> .
however, the stability order in  <cit> , referring to Δg <dig> values of mismatched trinucleotide duplexes, is considering absolute stability parameters, whereas  <cit>  and our study consider mismatch discrimination with the corresponding pm binding affinity as a reference level.
therefore, the comparability with the rna/dna stability order in  <cit>  is limited.
a recent work on the impact of single base mms in rna-interference  – allele-specific gene silencing experiments  <cit>  – is interesting in the context of this study, since here the sequence recognition is based on base-pairing between the guide strand  and a complementary mrna.
schwarz et al.
have shown that among all mm-types incorporated at position  <dig> of the guide strand  purine-purine mms resulted in the least silencing of gene activity , whereas u·g, c·u and u·u mismatches resulted in a very efficient gene silencing .
it is assumed that purine-purine mms strongly interfere with the formation of an a-form helix between the guide strand and the target mrna  <cit> .
this appears to be in accordance with the findings of pozhitkov et al.
on rna/dna mm discrimination.
however, the inferred rna/rna mismatch stability order  is not normalized with the corresponding pm stabilities, but rather reflects the absolute impact of the mm base pairs in a given duplex sequence and cannot be easily compared to our study and to  <cit> .
CONCLUSIONS
we performed a comprehensive, array-based study on the influence of point defects on the binding affinity of oligonucleotide duplexes.
contrary to previous studies by others, we have employed well-defined hybridization conditions by using short, end-labeled oligonucleotide target sequences  and can therefore exclude that target secondary structure, steric hindrance, labeling or competitive effects are relevant for an explanation of the observed results.
in our microarray-based hybridization assays the binding affinity of mispaired duplexes is dominated by the influence of defect position.
the influence of the defect-type is about half in magnitude, when compared to defect-position.
there is also an influence of the neighboring sequence, which has farther reach than the defect next neighbor.
although this long reach interaction must somehow be related to the base stacking energies, we did not find a simple description.
we attribute so far unexplained long range effects, in particular a trough-shaped position dependence, to molecular dynamics.
we propose a molecular zipping mechanism as a suitable explanation.
zipping agrees well with the observation that group ii bulges  have stronger hybridization signals than expected from previous data.
experimentally, it is not completely clear, whether the strong positional influence on oligonucleotide binding affinity is restricted to surface-hybridization or if it is also relevant for solution-phase hybridization .
the comparison to other related work  <cit> , however, shows significant differences in the mm-type dependence of duplex binding affinities.
our comparative analysis of the impact of point defects on the binding affinity of dna/dna and rna/dna duplexes reveals that purine-purine mms are more destabilizing in the latter.
this may explain some discrepancies in the literature.
the use of dna microarrays enables a detailed investigation of oligonucleotide duplex binding affinities producing a wealth of data in simple experiments.
we demonstrate that important aspects  about the impact of point defects on oligonucleotide duplex binding affinities are not yet understood.
our results from simple, controlled experiments agree well with results from extracting data from complex dna target mixtures  <cit> .
this shows that dna hybridization on surfaces can be reproducible and quantitatively significant.
deviations from the behavior, which we describe here, are observed in microarray experiments and they must be due to complexity of dna target mixtures.

 dna microarray technology relies on the highly specific binding affinity of surface-tethered dna probe sequences to complementary target sequences. nucleic acid hybridization, the sequential base pairing between complementary probe and target strands, results in the formation of stable double-stranded duplexes. in microarray hybridization assays single-stranded nucleic acid targets – contained in a complex mixture of diffierent target sequences in solution – freely diffuse over the surface-tethered probes until they are captured by a complementary probe. target strands often carry fluorescent dye labels to enable quantitative detection of the individual target species. hybridized targets can be identified by the position of the corresponding microarray features within the regular grid of the dna microarray. in dna microarray applications, along with a high binding affinity, a high specificity of probe-target hybridization is required to discriminate between sometimes very similar homologous sequences. binding specificity is particularly important in genotyping applications where single nucleotide polymorphisms, genetic variations of single bases, are concerned. snps determine genetic individuality, but also predisposition to a variety of genetic diseases, response to drugs, pathogens, chemicals and other agents. snps are of great interest not only for genetic research but also for medical diagnostics and therapy. snps and point-mutations can be detected by means of relatively short to mer probes: already a single mismatched base pair can result in a significant decrease of the duplex binding affinity with respect to the corresponding perfect matching duplex. the binding affinity of mismatched duplexes – in bulk solution – is commonly predicted on the basis of the nearest-neighbor model. a recent study by pozhitkov et al. revealed a poor correlation between predicted duplex binding affinities and actual hybridization signal intensities implying that the thermodynamic properties of oligonucleotide hybridization on dna microarrays are by far not understood. in dna microarray experiments the binding affinity of mismatched oligonucleotide duplexes is governed not just by nearest-neighbor parameters – as in solution-phase hybridization – but mainly by the position of the defect. furthermore, the secondary structure of the long target strands and various surface effects have a significant influence on the microarray binding affinity. our study is a comprehensive approach to understand how point defects affect the hybridization of fluorescently labeled oligonucleotide targets to surface-bound oligonucleotide probes. rather than previous work on single base mms, which has been conducted with complex target mixtures either from pcr products or in vitro transcripts, we employ short, end-labeled oligonucleotide targets, thus avoiding labeling and steric hindrance related effects. in order to avoid competitive binding we perform each hybridization assay with a single target sequence. oligonucleotide target sequences were chosen to minimize secondary structures and any related influence on the hybridization signal. in particular we investigated differences between the impact of defects on dna/dna and analogue rna/dna duplexes. fluorescently labeled dna and rna target oligonucleotides. dna chips were fabricated by light-directed in situ synthesis with a digital micromirror device based maskless synthesis apparatus developed in our laboratory. sets of probe sequences were derived from probe sequence motifs by systematic variation of defect type and defect position including all single base mismatches, insertions and deletions. the design of the hybridization experiments enables discrimination between the strong influence of defect position and the more subtle defect-type and sequence related factors. after the current article was submitted, we became aware of further related studies in this area. suzuki et al. performed hybridization on custom nimbleexpress™ arrays to investigate the influence of the probe length and mismatch position on single base mm discrimination. fish et al. performed a direct comparison between hybridization signals from spotted microarrays and measured thermodynamic melting parameters. they report a linear relation between the duplex free energy and the microarray hybridization intensity. the focus of the present paper is on the impact of various defect types on the hybridization signal.  our microarray hybridization experiments performed in this study provide quantitative information on the binding affinity of individual mismatched duplexes by means of the hybridization signal intensity. since the absolute hybridization signal intensities of the different sequence motifs employed in this study are subject to a large variation we compare the mm hybridization signals with the corresponding pm hybridization signals. our experiments – experimental details are explained in the methods section – provide a measure for the mismatch discrimination with respect to the corresponding pm binding affinity, rather than an absolute measure for the mm binding affinity. the discrimination between the hybridization affinity of point-mutated probes and corresponding perfect matching probes depends on the stability of the particular probe sequence. in agreement with we observed that the mer probes are less discriminative with respect to point defects than the shorter mer probes. discrimination is also reduced for sequence motifs stabilized by a higher cg-content. mm defect position and hybridization affinity the "defect profile" plots show that the dominant parameter determining oligonucleotide probe-target-affinity – on the microarray surface – is the position of the defect. a moving average evidences a trough-like "mean profile" curve. a parabolic fit can provide a reasonable approximation for the average position dependence obtained from a large number of different sequence motifs. for mer duplexes a single base mismatch in the center typically results in 40% of the pm hybridization signal. however, for individual sequence motifs we found sequence-dependent deviations from the simple position dependence. the raw signal intensities and probe/target sequences of the experiment are given in additional file influence of the mismatch type in dna/dna duplexes in the following we use the notation of the mismatch base pair x·y consisting of the mismatched base x in the probe sequence and the base y in the target sequence. to investigate how the particular mm-types x·y affect duplex stability we measured probe-target-affinities for different sequence motifs. microarray hybridization experiments with single base mismatch probe sets as well as the extraction of their hybridization signals, which reflect duplex stability, are described in more detail in the methods section. owing to the limited number of available target oligonucleotides we restricted base substitutions to the probe sequences. the pm hybridization signals of the different mer sequence motifs display a strong variation. since the relative hybridization signal intensities within the individual probe sets are largely unaffected by this variation, we normalize the "defect profiles" by division with their standard deviation. the resulting database comprising normalized hybridization signals from about different single mm probe sequences, enables categorization of the binding affinities according to the mismatch type. for statistical analysis of mm type and nearest-neighbor influences the superposed positional influence needs to be eliminated by subtraction of the mean profile. the resulting position-independent defect profile consisting of influences of defect type and defect neighborhood only is shown in fig. 2b. the boxplot representation of this data in fig. demonstrates that mm-types affecting c·g base pairs systematically have lower median hybridization signal values than mm-types affecting a·t base pairs. we compared the mm-type related hybridization signal deviations δimp from the mean mm profiles to predicted gibbs free energy differences δΔg37∘=Δg37mm∘−Δg37pm∘ between mm and corresponding pm duplexes. δΔg37∘ were determined from mismatch nearest-neighbor thermodynamic parameters. our analysis indicates a decreasing trend of the δimp values with increasing δΔg37∘. moreover, we observed that single base mismatches with two a·t flanking base pairs tend to provide a better mismatch discrimination than mismatches flanked by two c·g base pairs. dna/dna single base bulge defects single base insertions and deletions owing to a surplus unpaired base in one of the two strands result in bulged duplexes. in our experiments the bulged base is located on the surface-bound probe strand, whereas in duplexes with single base deletions the bulge is on the target strand. we discovered that on average the positional dependence of the insertion and deletion defect profiles is very similar to the positional dependence of mismatch defect profiles. within one and the same individual defect profile, single base bulge defects originating from single base insertions or deletions display the same positional dependence as single base mismatch defects, qualitatively as well as quantitatively. on average single base insertion probes provide increased hybridization signals when compared to mm probes or single base deletions. besides the significantly increased hybridization signals of group ii insertions, this is due to the reduced number of binding base pairs in the mismatched duplexes. in single base insertions no binding base pair is substituted, but we see that the influence of the inserted base clearly depends on its neighbor. the individual curves show deviations from the mean profile, hybridization signals can be significantly increased over several consecutive defect positions. in particular base insertions next to identical bases result in systematically increased binding affinities – in comparison to insertions of non-identical bases. group ii bulges located near the center of mer probes often show hybridization signals with a similar intensity as the corresponding pm probe. a statistical analysis with a large dataset comprising hybridization signal data from different 20– mer probes indicates the general validity of the result. interestingly, systematically increased hybridization signals have also been observed for certain group i bulges: for g-insertions next to a t we frequently find increased binding affinities similar to that of group ii bulges. we further analyzed the degree of correlation between the binding affinities of probes with different insertion bases x and y: a clear correlation appears between the hybridization signals of probes with t- and g-insertions, and also, though less distinct, between a- and c-insertions. in contrast, we observed an anti-correlation between g- and a-insertions. dna/dna versus dna/rna mismatch and bulged hybridization to investigate if the above results from dna/dna hybridization also apply to hybridization of rna/dna duplexes we performed a direct comparison employing dna targets and corresponding rna targets on the same microarray. we observed that mm discrimination in rna/dna duplexes is similar to mm discrimination in dna/dna duplexes. a statistical analysis reveals, however, that purine-purine mms are somewhat less stable in rna/dna duplexes than in dna/dna duplexes. the most significant differences between rna/dna and dna/dna mms are observed for the mm-types g·a and a·g and for the mm-type t·g. a presumed destabilizing effect of purine-purine mms in the ranking order of rna/dna mismatch discrimination is superposed to the affected base pair effect, which is very similarly, also observed in dna/dna hybridization. for bulged duplexes we did not observe significant defect-type specific differences between rna/dna and dna/dna hybridization. the hybridization signal and sequence data from the microarray hybridization experiment are provided in additional file single base insertion, deletion and mismatch defects in comparison defect profiles for mms and base bulges exhibit a very similar quantitative influence from defect position in dna/dna as well as in dna/rna complexes. for individual sequences the mean trough-shaped profile can be altered: fig. shows deformations of the trough-like profile on scales much larger than the size of a base pair. single base mm discrimination also depends on the type of mm base pair and the corresponding pm base pair. hybridization signals of mms originating from c·g base pairs are about 25% smaller than for mms from a·t base pairs. single base deletions affecting c·g base pairs result in about 30% smaller hybridization signals than deletions affecting a·t base pairs. the deletion profile in fig. shows that the local ups and downs of the profile curve correlate with deletions affecting either a·t or c·g base pairs. thus, for mms and single base deletions it is the type of base pair affected by the point-mutation, which determines the impact on hybridization affinity to an important degree, however, it is still less important than defect-position. we also observe a noticeable influence of the next-neighbor bases of the mismatch. discussion dominating influence of defect position we observe that defects located in the center of the oligonucleotide duplexes are significantly more destabilizing than defects at the ends. similar influence of the mm position has been reported previously from other microarray based studies, and also – although sparsely – from hybridization experiments in solution. the limited data in solution may be due to the technical difficulty of studying a large number of different probes. quantitatively, in accordance with we have identified mm position as the strongest influential factor on the hybridization signal, when compared to mm-type and nearest neighbor effects. the well-established two-state nearest-neighbor model, which has proved to be reliable for the prediction of duplex stabilities in solution-phase, does not regard the position of the nn pairs. we propose that a model for the prediction of microarray binding affinities should also include the position of the nn pairs – in particular in case of mismatched nn pairs. affinity models for microarray hybridization considering a positional dependence of the nearest-neighbor parameters have been previously discussed in. we observe a very similar position dependence for single base bulge defects as for single base mismatches. also, the magnitudes of the impacts of the mms and base bulges on the hybridization signal are very similar. this consistency suggests a common origin of the positional influence, independent of defect type. sterical crowding at the surface, as suggested by peterson et al., can in principle reduce the accessibility of the probe surface-bound 3'-ends and can thus decrease the impact of defects located near this end. however, in our case we observe largely symmetrical intensity profiles with respect to both ends of the probes. focusing on individual probe sequence motifs we observe, that the positional influence does not only depend on the defect-to-end distance, but also has a sequence-dependent contribution. this indicates that the impact of a defect also depends on the stability of the local sequence environment. since there are no long range molecular forces, we infer that the molecular dynamics must play a role, effects like breathing bubbles or zipping could be at the origin. this influence of the duplex sequence and the observed symmetry of the defect positional influence with respect to the duplex ends suggest that end-domain opening must be suspected to be a key mechanism for understanding the influence of defect position on duplex stability. influence of the mm-type removing the positional influence in our data, we see that single-base mms introduced at the site of a c·g base pair result in a larger decrease of the hybridization signal than mm defects affecting a·t base pairs. the same applies for single base deletions. these experimental results, in accordance with nearest-neighbor thermodynamic parameters for watson-crick base pairs, mainly reflect the increased base stacking and hydrogen bonding interactions of c·g base pairs. we observe a positive correlation between the experimentally determined single base mismatch discrimination and predicted free energy increments δΔg37∘ on the basis of the nearest-neighbor model – for details see additional file a similar correlation hybridization signal values and δΔg37∘) has been reported previously in. we emphasize the good correlation between our dna/dna mm stability order and the corresponding results of wick et al. hybridization signal values in fig. 5a in ). a major difference, however, occurs for the mm-pair g·g, which is the least stable in our study. wick found g·g to be one of the most stable mms. interestingly, however, pozhitkov et al. – in accordance with our results -identified g·g as one of the least stable mm-types. our direct comparison between dna/dna and rna/dna hybridization on microarrays reveals – for rna/dna duplexes – an increased destabilization of purine-purine mismatches, with respect to other mm types. an explanatory approach for the observed differences between dna/dna and rna/dna binding affinities is, that purine-purine mms cause larger steric hindrance in the a-form hybrid duplexes than in the b-form dna/dna duplexes. in contrast to we did not observe that purine-purine mismatches in rna/dna duplexes are, in absolute terms, more discriminative than other mm-types. increased stability of group ii single base bulges we observe significantly increased hybridization signals of single- base insertion defects in which the insertion base is placed next to a like-base. our investigation shows that the difference between group i and group ii binding affinities δibulge is distinctly larger than the defect-type related variation of binding affinities δimm. for comparison, the free energy differences among the mm trinucleotide duplexes abc/a¯xc¯ and abc/a¯yc¯ span the range δΔgmm = to kcal/mol. the increased stability of group ii bulges in comparison with group i bulges has been investigated previously in solution rather than on microarrays. according to ke and wartell the increased stability of group ii bulges originates from positional degeneracy of the base bulge. additional conformational freedom, entailing higher entropy, results in lowered duplex free energy. according to zhu et al. position degeneracy accounts for an average stabilization of - to - kcal/mol for a two-position degeneracy. znosko et al. reported group ii duplexes to be on average δΔg = - kcal/mol more stable than group i duplexes. the latter value matches better our observation of group ii hybridization close to the perfect match hybridization signal. for explanation of the large binding affinity of group ii duplexes we propose the following mechanism based on the molecular zipper model: even in thermal equilibrium due to thermal excitation, zipping as well as unzipping occur. the extend of the end-domain denaturation of the duplex, which is described by a random walk, may finally result in complete dissociation of the duplex. the binding affinity is determined by the ratio knuc/kdiss between the nucleation rate knuc and the duplex dissociation rate kdiss. we consider that a defect does not have an important influence on the unzipping, since the defect does not present a barrier for the process. for closing of the strands, however, the situation is different. the surplus base must act as a kinetic barrier, interrupting the rapid zipping of the duplex. the 1-nt frameshift between the complementary strands, owing to the unpaired bulge base prevents closing beyond the defect and results in a partially zipped, and correspondingly weakly-bound, duplex. duplex closure can only progress if the interfering surplus base is giving way, thus allowing the subsequent base to form a watson-crick base pair with the complementary base in the target strand. from this point zipping can progress rapidly. therefore compared to watson-crick nearest-neighbor pairs, a base bulge has a decreased ratio of zipping/unzipping-rates k+/k- and thus favors unzipping of the duplex. for group ii bulges the k+/k- ratio at the defect site is increased with respect to group i bulges: in case of a group ii bulge there is an increased probability that any of the degenerate bases makes way while simultaneously the subsequent base forms a base pair. this is due to the increased number of possible molecular conformations, which can lead to continuation of the zipping. then, as the frameshift is compensated, the rapid zipping to complete the duplex occurs. since the nucleation rate knuc of group i and group ii duplexes may be assumed to be the same, the binding affinity of group ii duplexes must be increased. previous studies – including rna/dna hybridization tautz and coworkers performed a mismatch study with mer oligonucleotide microarrays fabricated by light-directed in situ synthesis with the geniom® one instrument. similar as in our study, they compared normalized hybridization signal intensities. however, an important difference between the experiments described in and our experiments is the use of in vitro transcribed rna targets originating from ribosomal rna. they observe a more pronounced destabilization by purine-purine mms compared to our results. a further study on the impact of mm stabilities in rna/dna duplexes, in solution rather than on a microarray surface, has been published by sugimoto et al.. as discussed in the destabilizing effect of purine-purine mms is not observed by sugimoto et al.. however, the stability order in, referring to Δg values of mismatched trinucleotide duplexes, is considering absolute stability parameters, whereas and our study consider mismatch discrimination with the corresponding pm binding affinity as a reference level. therefore, the comparability with the rna/dna stability order in is limited. a recent work on the impact of single base mms in rna-interference – allele-specific gene silencing experiments – is interesting in the context of this study, since here the sequence recognition is based on base-pairing between the guide strand and a complementary mrna. schwarz et al. have shown that among all mm-types incorporated at position of the guide strand purine-purine mms resulted in the least silencing of gene activity, whereas u·g, c·u and u·u mismatches resulted in a very efficient gene silencing. it is assumed that purine-purine mms strongly interfere with the formation of an a-form helix between the guide strand and the target mrna. this appears to be in accordance with the findings of pozhitkov et al. on rna/dna mm discrimination. however, the inferred rna/rna mismatch stability order is not normalized with the corresponding pm stabilities, but rather reflects the absolute impact of the mm base pairs in a given duplex sequence and cannot be easily compared to our study and to.  we performed a comprehensive, array-based study on the influence of point defects on the binding affinity of oligonucleotide duplexes. contrary to previous studies by others, we have employed well-defined hybridization conditions by using short, end-labeled oligonucleotide target sequences and can therefore exclude that target secondary structure, steric hindrance, labeling or competitive effects are relevant for an explanation of the observed results. in our microarray-based hybridization assays the binding affinity of mispaired duplexes is dominated by the influence of defect position. the influence of the defect-type is about half in magnitude, when compared to defect-position. there is also an influence of the neighboring sequence, which has farther reach than the defect next neighbor. although this long reach interaction must somehow be related to the base stacking energies, we did not find a simple description. we attribute so far unexplained long range effects, in particular a trough-shaped position dependence, to molecular dynamics. we propose a molecular zipping mechanism as a suitable explanation. zipping agrees well with the observation that group ii bulges have stronger hybridization signals than expected from previous data. experimentally, it is not completely clear, whether the strong positional influence on oligonucleotide binding affinity is restricted to surface-hybridization or if it is also relevant for solution-phase hybridization. the comparison to other related work, however, shows significant differences in the mm-type dependence of duplex binding affinities. our comparative analysis of the impact of point defects on the binding affinity of dna/dna and rna/dna duplexes reveals that purine-purine mms are more destabilizing in the latter. this may explain some discrepancies in the literature. the use of dna microarrays enables a detailed investigation of oligonucleotide duplex binding affinities producing a wealth of data in simple experiments. we demonstrate that important aspects about the impact of point defects on oligonucleotide duplex binding affinities are not yet understood. our results from simple, controlled experiments agree well with results from extracting data from complex dna target mixtures. this shows that dna hybridization on surfaces can be reproducible and quantitatively significant. deviations from the behavior, which we describe here, are observed in microarray experiments and they must be due to complexity of dna target mixtures. 
the influence of single base bulges and single base mms depends predominantly on position – it is largest in the middle of the strand. the position-dependent influence of base bulges is very similar to that of single base mms, however certain bulges give rise to an unexpectedly high binding affinity. we performed microarray hybridization experiments with short to mer target and probe lengths in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. direct comparison between binding affinities of dna/dna and rna/dna duplexes shows, that rna/dna purine-purine mms are more discriminating than corresponding dna/dna mms. we have shown that dna microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. in dna/dna mm discrimination the affected base pair is the pertinent parameter. in order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. we have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. besides the defect type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. in order to explain our observations we propose dna molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. microarrays usually address complex mixtures of dna targets.
 the high binding specificity of short to mer oligonucleotide probes enables single base mismatch discrimination and thus provides the basis for genotyping and resequencing microarray applications. recent experiments indicate that the underlying principles governing dna microarray hybridization – and in particular mm discrimination – are not completely understood. microarrays usually address complex mixtures of dna targets. in order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.  we performed microarray hybridization experiments with short to mer target and probe lengths in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. the influence of single base bulges and single base mms depends predominantly on position – it is largest in the middle of the strand. the position-dependent influence of base bulges is very similar to that of single base mms, however certain bulges give rise to an unexpectedly high binding affinity. besides the defect type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. direct comparison between binding affinities of dna/dna and rna/dna duplexes shows, that rna/dna purine-purine mms are more discriminating than corresponding dna/dna mms. in dna/dna mm discrimination the affected base pair is the pertinent parameter. we attribute these differences to the different structures of the duplexes.  we have shown that dna microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. we have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. in order to explain our observations we propose dna molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. 