Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100 mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.     

The stability of intramolecular DNA quadruplexes with extended loops forming inter- and intra-loop duplexes

We have examined the formation of intramolecular quadruplex DNA structures in which the loops have been extended so as to generate short DNA duplexes. Fluorescence melting and DNase I cleavage studies show that duplexes can be formed within each loop, but that duplexes between the loops are not stable.     

Coordination-driven inversion of handedness in ligand-modified PNA

Peptide nucleic acid (PNA) is a synthetic analogue of DNA, which has the same nucleobases as DNA but typically has a backbone based on aminoethyl glycine (Aeg). PNA forms duplexes by Watson Crick hybridization. The Aeg-based PNA duplexes adopt a chiral helical structure but do not have a preferred handedness because they do not contain a chiral center. An L-lysine situated at the C-end of one or both strands of a PNA duplex causes the duplex to preferably adopt a left-handed structure. We have introduced into the PNA duplexes both a C-terminal L-lysine and one or two PNA monomers that have a γ-(S)-methyl-aminoethyl glycine backbone, which is known to induce a preference for a right-handed structure. Indeed, we found that in these duplexes the γ-methyl monomer exerts the dominant chiral induction effect causing the duplexes to adopt a right-handed structure. The chiral PNA monomer had a 2,2':6',2'-terpyridine (Tpy) ligand instead of a nucleobase and PNA duplexes that contained one or two Tpys formed [Cu(Tpy)(2)](2+) complexes in the presence of Cu(2+). The CD spectroscopy studies showed that these metal-coordinated duplexes were right-handed due to the chiral induction effect exerted by the S-Tpy PNA monomer(s) except for the cases when the [Cu(Tpy)(2)](2+) complex was formed with Tpy ligands from two different PNA duplexes. In the latter case, the metal complex bridged the two PNA duplexes and the duplexes were left-handed. The results of this study show that the preferred handedness of a ligand-modified PNA can be switched as a consequence of metal coordination to the ligand. This finding could be used as a tool in the design of functional nucleic-acid based nanostructures.     

An unusual mode of DNA duplex association: Watson-Crick interaction of all-purine deoxyribonucleic acids

Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.     

DNA stability in the gas versus solution phases: A systematic study of thirty-one duplexes with varying length, sequence, and charge level

We report herein a systematic mass spectrometric study of a series of thirty-one non-self-complementary, matched, DNA duplexes ranging in size from 5- to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same Tm yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O6-methylguanine causes mutations.     

Synthesis of oligodiaminosaccharides having α-glycoside bonds and their interactions with oligonucleotide duplexes

Syntheses of the novel oligodiaminosaccharides, α-(1→4)-linked-2,6-diamino-2,6-dideoxy-D-glucopyranose oligomers, and their interactions with nucleic acid duplexes DNA-DNA, RNA-RNA, and DNA-RNA are described. Monomers to tetramers of oligodiaminoglucose derivatives having α-glycosyl bonds were successfully synthesized using a chain elongation cycle including glycosylation reactions of a 6-phthalimide glycosyl donor. UV melting experiments for a variety of nucleic acid duplexes in the absence and presence of the oligodiaminosaccharides were performed. The synthesized oligodiaminosaccharides exhibited notable thermodynamic stabilization effects on A-type RNA-RNA and DNA-RNA duplexes, whereas B-type DNA-DNA duplexes were not stabilized by the synthesized oligodiaminosaccharides. Among the oligodiaminosaccharides, the tetramer exhibited the highest ability to stabilize A-type duplexes, and the increase in T(m) values induced by the tetramer were higher than those induced by neomycin B and tobramycin, which are known aminoglycosides having ability to bind and stabilize a variety of RNA molecules. CD spectrometry experiments revealed that the oligodiaminosaccharides caused small structural changes in RNA-RNA duplexes, whereas no appreciable changes were observed in the structure of DNA-DNA duplexes. ITC (isothermal titration calorimetry) experiments demonstrated that the amount of heat generated by the interaction between RNA-RNA duplexes and the tetradiaminosaccharides was approximately double that generated by that between DNA-DNA duplexes and the tetradiaminosaccharides. These results strongly suggested the existence of an A-type nucleic acid specific-binding mode of the oligodiaminosaccharides, which bind to these duplexes and cause small structural changes.     

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4'-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect. The aglycone skeletons and other hydroxyl substitutions on the aglycone also have an effect on the fractions of bound DNA. Upon collision-induced dissociation, the complexes containing flavonoid aglycones underwent the predominant ejection of a neutral ligand molecule, suggesting an intercalative DNA-binding mode. However, for complexes containing flavonoid glycosides, the loss of nucleobase increased to different extents, indicating a stronger binding or different binding mode. The results may provide not only a deeper insight into the DNA-binding properties of flavonoids but also a useful guideline for the design of efficient DNA-binding agents for chemotherapy.     

Isostable DNA

The high fidelity detection of multiple DNA sequences in multiplex assays calls for duplexes whose stability is independent of sequence (isostable DNA), forming under universally stringent conditions. Nature did not evolve DNA to form isostable duplexes. Here we report how probe strands can be modified so that an all-A/T target strand is bound with the same or slightly higher affinity than the corresponding all-G/C strand with the same sequence of purines and pyrimidines. We refer to these probes that feature covalently attached ligands as "decorated nucleic acids". Caps, intercalators, and locks were used to stabilize A/T duplexes, and N4-ethylcytosine residues were employed to tune down the stability of G/C duplexes without significantly affecting base pairing selectivity. Near-isostability was demonstrated in solution and on microarrays of high and low density. Further, it is shown that hybridization results involving decorated probes on microarrays can be predicted on the basis of thermodynamic data for duplex formation in solution. Predictable formation of isostable DNA not only benefits microarrays for gene expression analysis and genotyping, but may also improve the sequence-specificity of other applications that rely on the massively parallel formation of Watson-Crick duplexes.     

Oligodeoxynucleotide Duplexes Containing (5′S)‐5′‐C‐Alkyl‐Modified 2′‐Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor‐Groove Enhance Duplex Stability?

A series of oligonucleotides containing (5′S)‐5′‐C‐butyl‐ and (5′S)‐5′‐C‐isopentyl‐substituted 2′‐deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl‐zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA‐duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I – III , Fig. 2) could experimentally be realized and their duplex‐forming properties analyzed by UV‐melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in T_m. We found that, within the proposed duplexes with more loosely packed alkyl groups (type‐ III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B‐DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type‐ II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type‐ III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl‐zipper formation presumably by loss of structured H₂O in the minor groove.     

Metal binding to ligand-containing peptide nucleic acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.     

Alternative strategy for adjusting the association specificity of hydrogen-bonded duplexes

A strategy for creating new association specificity of hydrogen-bonded duplexes by varying the spacings between neighboring hydrogen bonds is described. Incorporation of naphthalene-based residues has provided oligoamide strands that pair into duplexes sharing the same H-bonding sequences (e.g., DDAA) but differing in the spacings between their intermolecular hydrogen bonds, leading to homo- or heteroduplexes. The ability to manipulate association-specificity as demonstrated by this work may be extended to other multiple hydrogen bonded systems, thereby further enhancing the diversity of multiple hydrogen-bonded association units for constructing supramolecular structures.     

Bicyclo[3.2.1]amide-DNA: a chiral,nonchiroselective base-pairing system

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleo-bases were attached through an extended, acyclic amide linker. Homobasic adenine-containing bca decamers form duplexes with complementary oligonucleotides containing bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA-containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association.     

Specific Induced Circular Dichroism and Enhanced B to Z Transitions of Duplexes Stabilized by Chromophore‐Linked Alkynylnucleoside Residues

We have developed an induced circular dichroism (ICD) probe with a chromophore‐linked alkynyldeoxyribose skeleton for analyzing higher‐order structures of DNA duplexes in the visible‐light region. When CG‐repeated oligonucleotides (ODNs) with the probe at their 5′ ends adopted Z‐form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B‐form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z‐form duplexes was found to appear only when the chromophores were attached at the 5′ ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.     

2-Aminopurine: a probe of structural dynamics and charge transfer in DNA and DNA:RNA hybrids

Spectroscopic techniques are employed to probe relationships between structural dynamics and charge transfer (CT) efficiency in DNA duplexes and DNA:RNA hybrids containing photoexcited 2-aminopurine (Ap). To better understand the variety of interactions and reactions, including CT, between Ap and DNA, the fluorescence behavior of Ap is investigated in a full series of redox-inactive as well as redox-active assemblies. Thus, Ap is developed as a dual reporter of structural dynamics and base-base CT reactions in nucleic acid duplexes. CD, NMR, and thermal denaturation profiles are consistent with the family of DNA duplexes adopting a distinct conformation versus the DNA:RNA hybrids. Fluorescence measurements establish that the d(A)-r(U) tract of the DNA:RNA hybrid exhibits enhanced structural flexibility relative to that of the d(A)-d(T) tract of the DNA duplexes. The yield of CT from either G or 7-deazaguanine (Z) to Ap in the assemblies was determined by comparing Ap emission in redox-active G- or Z-containing duplexes to otherwise identical duplexes in which the G or Z is replaced by inosine (I), the redox-inactive nucleoside analogue. Investigations of CT not only demonstrate efficient intrastrand base-base CT in the DNA:RNA hybrids but also reveal a distance dependence of CT yield that is more shallow through the d(A)-r(U) bridge of the A-form DNA:RNA hybrids than through the d(A)-d(T) bridge of the B-form DNA duplexes. The shallow distance dependence of intrastrand CT in DNA:RNA hybrids correlates with the increased conformational flexibility of bases within the hybrid duplexes. Measurements of interstrand base-base CT provide another means to distinguish between the A- and B-form helices. Significantly, in the A-form DNA:RNA hybrids, a similar distance dependence is obtained for inter- and intrastrand reactions, while, in B-DNA, a more shallow distance dependence is evident with interstrand CT reactions. These observations are consistent with evaluations of intra- and interstrand base overlap in A- versus B-form duplexes. Overall, these data underscore the sensitivity of CT chemistry to nucleic acid structure and structural dynamics.     

Charge transfer through modified peptide nucleic acids

We studied the charge transfer properties of bipyridine-modified peptide nucleic acid (PNA) in the absence and presence of Zn(II). Characterization of the PNA in solution showed that Zn(II) interacts with the bipyridine ligands, but the stability of the duplexes was not affected significantly by the binding of Zn(II). The charge transfer properties of these molecules were examined by electrochemistry for self-assembled monolayers of ferrocene-terminated PNAs and by conductive probe atomic force microscopy for cysteine-terminated PNAs. Both electrochemical and single molecular studies showed that the bipyridine modification and Zn(II) binding do not affect significantly the charge transfer of the PNA duplexes.     

Comparative stability determination of oligonucleotide duplexes in gas and solution phase

The relative stability of RNA duplexes were determined in both solution and gas phase. Solution stability as determined by a spectrophotometric method indicated that the Watson-Crick duplexes were more stable than duplexes containing one GA mismatch or two tandem GA mismatches. Gas phase stability was determined using ESI-MS through variation of the collision energy in an ion trap. Stability curves similar to the melting curves obtained in solution were observed. The relative stability in gas phase differed, however, from that in solution. The duplexes with two tandem GA mismatches were found to be more stable than the Watson-Crick and single GA mismatch duplexes. The different trends observed in solution versus gas phase can be attributed to the primary means of interaction. In solution, stacking is expected to be the dominant interaction mode. In the gas phase, hydrogen bonding is expected to be the dominant interaction mode. Duplexes with tandem GA mismatches have the potential to undergo additional hydrogen bonding relative to the other duplexes which could account for their increased stability in the gas phase.     

DNA sequence context and multiplex hybridization reactions: melting studies of heteromorphic duplex DNA complexes

Heteromorphic hybrid duplex DNA complexes are duplex states, other than perfectly matched duplexes, that can form when single strands comprising several different perfectly matched duplexes are simultaneously present in solution. Such cross-hybridization "side reactions" are of particular nuisance in multiplex reaction schemes, where many strands are designed to hybridize in parallel fashion with only their corresponding perfect complement strand. Relative to the perfect match duplexes, the sequence dependent features of these heteromorphic duplex states and their thermodynamic stability are an important consideration for multiplex hybridization reaction design. We have measured absorbance versus temperature melting curves and performed differential scanning calorimetry measurements on various mixtures of eight different 24 base single strands. When perfect complementary pairs of strands are mixed in single reactions, four perfectly matched duplexes form. When mixtures of strands that are not perfectly matched are prepared and analyzed, melting transitions for cross-hybridization are observed along with significant hyperchromicity changes. This is indicative of a melting hybrid, heteromorphic duplex states formed from two nonperfectly matched strands. In addition, when both the perfectly matched and noncomplementary strands are mixed together (in multiplex hybridization reactions) at molar ratios of 1:1, 3:1, and 1:3, evidence of perfect duplex and heteromorphic duplex complexes is found in all cases. A new analytical tool for considering heterogeneous, duplex complexes in multiplex hybridization mixtures is presented and employed to interpret the acquired melting data.     

How do size-expanded DNA nucleobases enhance duplex stability? Computational analysis of the hydrogen-bonding and stacking ability of xDNA bases

Computational chemistry (B3LYP, MP2) is used to study the properties of size-expanded DNA nucleobases generated by inserting a benzene spacer into the natural nucleobases. Although the addition of the spacer does not significantly affect the hydrogen-bonding properties of natural nucleobases, the orientation of the base about the glycosidic bond necessary for Watson-Crick binding is destabilized, which could have implications for the selectivity of expanded bases, as well as the stability of expanded duplexes. Consideration of the (stacked) binding energies in the preferred relative orientation of natural and expanded nucleobases aligned according to their centers of mass reveals that the stacking within natural dimers can be increased by up to 50% upon expansion of one nucleobase and up to 90% upon expansion of two nucleobases. The implications of these findings to the stability of expanded duplexes were revealed by considering simplified models of natural and mixed duplexes composed of four nucleobases. Although intra- and interstrand interactions within double helices are typically less than those predicted when nucleobases are stacked according to their centers of mass, some nucleobases utilize their full stacking potential within double helices, where both intra- and interstrand interactions can be significant. Most importantly, increasing the size of nucleobases within the duplex significantly increases both intra- and interstrand stacking interactions. Specifically, some interactions are double the magnitude of the corresponding intrastrand interactions in natural helices, and even greater increases in interstrand interactions are sometimes found. Thus, our work suggests that mixed duplexes composed of natural bases hydrogen bound to expanded bases may exploit the increase in the inherent stacking ability of the expanded bases in more than one way and thereby afford duplexes with greater stability than natural DNA.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu(2+). Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu(2+), duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu(2+) ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu(2+) of the 1:2 Cu(2+):Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ(2)] moieties that are similar to the corresponding square planar synthetic complex formed between Cu(2+) and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal l-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.