Expanding the repertoire of heterocycle ring pairs for programmable minor groove DNA recognition

The discrimination of the four Watson-Crick base pairs by minor groove DNA-binding polyamides have been attributed to the specificity of three five-membered aromatic amino acid subunits, 1-methyl-1H-imidazole (Im), 1-methyl-1H-pyrrole (Py), and 3-hydroxy-1H-pyrrole (Hp) paired four different ways. The search for additional ring pairs that demonstrate DNA-sequence specificity has led us to a new class of 6-5 fused bicycle rings as minor groove recognition elements. The affinities and specificities of the hydroxybenzimidazole/pyrrole (Hz/Py) and hydroxybenzimidazole/benzimidazole (Hz/Bi) pairs for each of the respective Watson-Crick base pairs within the sequence context 5'-TGGXCA-3' (X = A, T, G, C) were measured by quantitative DNaseI footprinting titrations. The Hz/Py and Hz/Bi distinguish T.A from A.T. Hairpin polyamides containing multiple Hz/Py pairs were examined and were shown to mimic the Hp/Py pair with regard to affinity and specificity. Therefore, the Hz/Py pair may be considered a second-generation replacement for the Hp/Py pair.     

On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides

Background: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (lm) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G·C and A·T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G·C base pairs.Results: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G·C and A·T base pairs in the central position. We observed that a G·C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A·T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im.Conclusions: An Im/Im pairing is energetically disfavored for the recognition of both A·T and G·C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.     

Programmable oligomers for minor groove DNA recognition

The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.     

Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing

This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, (Py)U and (Py)C, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a (Py)U/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing (Py)U/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched (Py)U/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a (Py)U/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, (Py)C, selectively emitted fluorescence when the base opposite (Py)C was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. (Py)U- and (Py)C-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.     

Sequence specific fluorescence detection of double strand DNA

Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide-fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of >/=1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide-TMR conjugate. Polyamide-fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson-Crick DNA base pair. A ranking of the specificity of three polyamide ring pairs-Py/Py, Im/Py, and Im/Im-was established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T.G base pair.     

Extending the language of DNA molecular recognition by polyamides: unexpected influence of imidazole and pyrrole arrangement on binding affinity and specificity

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity has been investigated using DNA melting (DeltaT(M)), circular dichroism (CD), and surface plasmon resonance (SPR) studies. SPR results obtained from a complete set of triheterocyclic polyamides show a dramatic difference in the affinity of f-ImPyIm for its cognate DNA (K(eq) = 1.9 x 10(8) M(-1)) and f-PyPyIm for its cognate DNA (K(eq) = 5.9 x 10(5) M(-1)), which could not have been anticipated prior to characterization of these compounds. Moreover, f-ImPyIm has a 10-fold greater affinity for CGCG than distamycin A has for its cognate, AATT. To understand this difference, the triamide dimers are divided into two structural groupings: central and terminal pairings. The four possible central pairings show decreasing selectivity and affinity for their respective cognate sequences: -ImPy > -PyPy- > -PyIm- approximately -ImIm-. These results extend the language of current design motifs for polyamide sequence recognition to include the use of "words" for recognizing two adjacent base pairs, rather than "letters" for binding to single base pairs. Thus, polyamides designed to target Watson-Crick base pairs should utilize the strength of -ImPy- and -PyPy- central pairings. The f/Im and f/Py terminal groups yielded no advantage for their respective C/G or T/A base pairs. The exception is with the -ImPy- central pairing, for which f/Im has a 10-fold greater affinity for C/G than f/Py has for T/A.     

Discrimination of A/T sequences in the minor groove of DNA within a cyclic polyamide motif

Eight-ring cyclic polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp) aromatic amino acids recognize predetermined six base pair sites in the minor groove of DNA. Two four-ring polyamide subunits linked by (R)-2,4-diaminobutyric acid [(R)H2Ngamma] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin polyamides, substitution of Hp/Py for Py/Py pairs enhances selectivity for T. A base pairs but compromises binding affinity for specific sequences. In an effort to enhance the binding properties of polyamides containing Hp/Py pairings, four eight ring cyclic polyamides were synthesized and analyzed on a DNA restriction fragment containing three 6-bp sites 5'-tAGNNCTt-3', where NN = AA, TA, or AT. Quantitative footprint titration experiments demonstrate that contiguous placement of Hp/Py pairs in cyclo-(gamma-ImPyPyPy-(R)H2Ngamma-ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5'-tAGAACTt-3' site (Ka = 7.5 x 10(7)M(-1)) relative to ImPyPyPy-(R)H2Ngamma-ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(gamma-ImHpPyPy-(R)H2Ngamma-ImHpPyPy-) (3) binds a 5'-tAGTACTt-3' site with an equilibrium association constant KA= 3.2 x 10(9)M(-1), representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)H2Ngamma-ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3'-stagger regulates specificity of cyclo-(gamma-ImPyHpPy-(R)H2Ngamma-ImPyHpPy-) (5) for the 5'-tAGATCTt-3' site (Ka = 7.5 x 10(7)M(-1)) threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)H2Ngamma-ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.     

碱基对在DNA双螺旋链上分离的自由能计算

DNA是大部分生物包括病毒的基因载体。DNA双螺旋链通过A＝T和G≡C两种碱基对编码实现对遗传信息的存储。碱基对中的相互作用对DNA双螺旋链的稳定性起到重要作用,直接关系到基因的复制和转录。当前研究中,我们构建了四组不同结构的DNA双螺旋链,进行了总共4.3 μs的分子动力学模拟。通过伞形取样技术计算了DNA双螺旋链中碱基对分离的自由能曲线,并从分子尺度细节和相互作用能对自由能曲线进行解析。在碱基对G≡C的自由能曲线(PMF-PGC)上观察到三个峰,通过监测氢键数目的变化发现分别对应于G≡C三个氢键的断裂;而在A＝T的自由能曲线(PMF-PAT)上只出现一个峰,说明A＝T的两个氢键在分离过程中几乎同时断裂。PMF-PGC的总能垒比PMF-PAT高,主要是因为G≡C比A＝T多一个氢键,更稳定。两条曲线的后段自由能仍然升高,而此时碱基对的氢键已断裂,这是DNA链骨架刚性所导致。我们还研究了碱基对稳定性受相邻碱基对的影响,发现邻近G≡C碱基对会增强A＝T的稳定性, C≡G会削弱A＝T的稳定性, T＝A对A＝T的影响较小。     

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.     

Photoreaction at 5'-(G/C)AA(Br)UT-3' sequence in duplex DNA: efficient generation of uracil-5-yl radical by charge transfer

The photoreactivities of 5-halouracil-containing DNA have widely been used for analysis of protein-DNA interactions and have recently been used for probing charge-transfer processes along DNA. Despite such practical usefulness, the detailed mechanisms of the photochemistry of 5-halouracil-containing DNA are not well understood. We recently discovered that photoirradiation of BrU-substituted DNA efficiently produced 2'-deoxyribonolactone at 5'-(G/C)AABrUBrU-3' and 5'-(G/C)ABrUBrU-3' sequences in duplex DNA. Using synthetic oligonucleotides, we found that similar photoreactivities were maintained at the 5'-(G/C)AABrUT-3' sequence, providing ribonolactone as a major product with concomitant release of adenine base. In this paper, the photoreactivities of various oligonucleotides possessing the 5'-BrUT-3' sequence were examined to elucidate the essential factors of this photoreaction. HPLC product analysis indicated that the yield of 2'-deoxyribonolactone largely depends on the ionization potential of the purine derivatives located 5'-upstream of 5'-BrUT-3', as well as the electron-donating ability of their pairing cytosine derivatives. Oligonucleotides that possess G in the complementary strand provided the ribonolactone with almost the same efficiency. These results clearly suggest that the photoinduced charge transfer from the G-5' upstream of 5'-BrUT-3' sequence, in the same strand and the complementary strand, initiates the reaction. To examine the role of intervening A/T base pair(s) between the G/C and the 5'-BrUT-3' sequence, the photoreactivities of a series of oligonucleotides with different numbers of intervening A/T base pairs were examined. The results revealed that the hotspot sequence consists of the electron-donating G/C base pair, the 5'-BrUT-3' sequence as an acceptor, and an appropriate number of A/T base pairs as a bridge for the charge-transfer process.     

Toward an Understanding of the Chemical Etiology for DNA Minor‐Groove Recognition by Polyamides

Crescent‐shaped polyamides composed of aromatic amino acids, i.e., 1‐methyl‐1H‐imidazole Im , 1‐methyl‐1H‐pyrrole Py , and 3‐hydroxy‐1H‐pyrrole Hp , bind in the minor groove of DNA as 2 : 1 and 1 : 1 ligand/DNA complexes. DNA‐Sequence specificity can be attributed to shape‐selective recognition and the unique corners or pairs of corners presented by each heterocycle(s) to the edges of the base pairs on the floor of the minor groove. Here we examine the relationship between heterocycle structure and DNA‐sequence specificity for a family of five‐membered aromatic amino acids. By means of quantitative DNase‐I footprinting, the recognition behavior of polyamides containing eight different aromatic amino acids, i.e., 1‐methyl‐1H‐pyrazole Pz , 1H‐pyrrole Nh , 5‐methylthiazole Nt , 4‐methylthiazole Th , 3‐methylthiophene Tn , thiophene Tp , 3‐hydroxythiophene Ht , and furan Fr , were compared with the polyamides containing the parent‐ring amino acids Py, Im , and Hp for their ability to discriminate between the four Watson? Crick base pairs in the DNA minor groove. Analysis of the data and molecular modeling showed that the geometry inherent to each heterocycle plays a significant role in the ability of polyamides to differentiate between DNA sequences. Binding appears sensitive to changes in curvature complementarity between the polyamide and DNA. The Tn / Py pair affords a modest 3‐fold discrimination of T?A vs. A?T and suggests that an S‐atom in the thiophene ring prefers to lie opposite T not A.     

Sequence‐Specific DNA Alkylation Targeting for Kras Codon 13 Mutation by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Hairpin N‐methylpyrrole‐N‐methylimidazole polyamide seco‐CBI conjugates 2 – 6 were designed for synthesis by Fmoc solid‐phase synthesis, and their DNA‐alkylating activities against the Kras codon 13 mutation were compared by high‐resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5′‐ACGTCACC A ‐3′ (site 2), including minor 1 bp‐mismatch alkylation against wild type 5′‐ACG C CACC A ‐3′ (site 3). Conjugate 6 , which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5′‐ACGTCACC A ‐3′ (site 2). The hairpin polyamide seco‐CBI conjugate 5 thus alkylates according to Dervan′s pairing rule with the pairing recognition which β/β pair targets T–A and A–T pairs. SPR and a computer‐minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.     

Λ-、△-[Rh（bpy）2chrysi]^3＋对含C：T错配的DNA序列识别作用的分子模拟研究

通过分子模拟方法研究了手性金属配合物[Rh（bpy）2Chrysi]^3＋（bpy=2，2’-bipyridineChrysi=5，6-chrysenequinonediimine）对包含C：T错配碱基对的B-DNA序列的识别作用．结合类似的针对含G：A错配的和正常的B-DNA序列的识别作用研究，发现配合物I-Rh（bpy）zChrysi]^3＋可以对错配B-DNA序列进行序列特异性的识别．能量对比计算结果表明，该经典插入识别作用倾向于在错配碱基对附近进行，其中△-[Rh（bpy）2chrysi]^3＋比其手性异构体更占优势．这同Barton教授工作组的实验结果是一致的．另外插入作用倾向于在错配序列中的正常双碱基对C3A4／G3T4（错配碱基对附近）中从小沟进行．与该配合物对含G：A错配的和正常的B-DNA序列的识别作用不同的是，对包含C：T错配碱基对的B-DNA序列的识别作用倾向于从小沟进行．这一点可能源于C：T碱基对结构的不同．     

Λ-、Δ-[Rh(bpy)2chrysi]3+对含C:T错配的DNA序列识别作用的分子模拟研究

通过分子模拟方法研究了手性金属配合物[Rh(bpy)2Chrysi]3 (bpy=2,2’- bipyridine;Chrysi=5,6-chrysenequinonediimine)对包含C:T错配碱基对的B-DNA序列的识别作用。结合类似的针对含G:A错配的和正常的B-DNA序列的识别作用研究,发现配合物[Rh(bpy)2Chrysi]3 可以对错配B-DNA序列进行序列特异性的识别．能量对比计算结果表明,该经典插入识别作用倾向于在错配碱基对附近进行,其中Δ-[Rh(bpy)2charysi]3 比其手性异构体更占优势．这同Barton教授工作组的实验结果是一致的。另外插入作用倾向于在错配序列中的正常双碱基对C3A4／G374(错配碱基对附近)中从小沟进行．与该配合物对含G:A错配的和正常的B-DNA序列的识别作用不同的是,对包含C:T错配碱基对的B-DNA序列的识别作用倾向于从小沟进行．这一点可能源于C:T碱基对结构的不同．     

Evaluation of Novel Third‐Strand Bases for the Recognition of a C⋅G Base Pair in the Parallel DNA Triple‐Helical Binding Motif

We describe the synthesis and the incorporation into oligonucleotides of the novel nucleoside building blocks 9, 10 , and 16 , carrying purine‐like double H‐bond‐acceptor bases. These base‐modified nucleosides were conceived to recognize selectively a cytosine⋅guanine (C⋅G) inversion site within a homopurine⋅homopyrimidine DNA duplex, when constituent of a DNA third strand designed to bind in the parallel binding motif. While building block 16 turned out to be incompatible with standard oligonucleotide‐synthesis conditions, UV/triplex melting experiments with third‐strand 15‐mers containing β‐D ‐nucleoside 6 (from 9 ) showed that recognition of the four natural Watson‐Crick base pairs follows the order G⋅C≈C⋅G>A⋅T>T⋅A. The recognition is sequence‐context sensitive, and G⋅C or C⋅G recognition does not involve protonated species of β‐D ‐nucleoside 6 . The data obtained fit (but do not prove) a structural model for C⋅G recognition via one conventional and one C−H⋅⋅⋅O H‐bond. The unexpected G⋅C recognition is best explained by third‐strand base intercalation. A comparison of the triplex binding properties of these new bases with those of 4‐deoxothymine (5‐methylpyrimidine‐2(1H)‐one, ^{4 H}T), previously shown to be C⋅G selective but energetically weak, is also described.     

Base pair recognition of the stereochemically alpha-substituted gamma-turn of pyrrole/imidazole hairpin polyamides

Recognition of the sequences 5'-NGCACA-3' (N = T, A, C, G) by pyrrole/imidazole polyamides with (R/S)-alpha-hydroxyl/alpha-amino-substituted gamma-aminobutyric acid as a gamma-turn was investigated. Four novel polyamides, 2, 3, 4, and 5, including (R)-alpha-hydroxyl-gamma-aminobutyric acid (gammaRO), (S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaSO), (R)-alpha,gamma-diaminobutyric acid (gammaRN), and (S)-alpha,gamma-diaminobutyric acid (gammaSN) residues, respectively, were synthesized, and their binding affinity to T.A, A.T, G.C, and C.G base pairs at turn position was studied by the surface plasmon resonance (SPR) technique. SPR data revealed that polyamide 3, AcImbetaImPy-gammaSO-ImPybetaPy-beta-Dp, with a gammaSO turn, possesses a marked binding preference for T.A over A.T with a 25-fold increase in specificity, despite low binding affinity relative to 2, with a gammaRO turn. Similarly, AcImbetaImPy-gammaSN-ImPybetaPy-beta-Dp (5), with a gammaSN-turn, gives rise to a 8.7-fold increase in specificity for T.A over A.T. Computer-assisted molecular modeling suggests that 3 binds more deeply in the minor groove of the T.A base pair relative to the A.T base pair, allowing hydrogen bonding to O2 of the thymine at the turn position, which explains the SPR results. These results suggest that gammaSO and gammaSN may function as T-recognition units at the turn position, as well as a gamma-turn in the discrimination of polyamides.     

Unnatural substrate repertoire of A, B, and X family DNA polymerases

As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examined the ability of five different polymerases to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five additional polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9(o)N-7, and the family X polymerase, human polymerase beta. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examined, suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is especially noteworthy due to strong mispair discrimination.     

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5'-AGTCAG-3' (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5'-AGTCAG-3' (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5'-AGTCCA-3' (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5'-TCACAA-3' (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervan's pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.     

Induced-fit recognition of DNA by small circular oligonucleotides

We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d with d, as well as with its two linear analogs d(GTCCCTCA) and d(CTCAGTCC). Here we report the NMR structural study of these complexes. Recognition between these oligonucleotides occurs through formation of four intermolecular Watson-Crick base pairs. The three-dimensional structure is stabilized by two tetrads, formed by facing the minor-groove side of the Watson-Crick base pairs. Overall, the structure is similar to those observed previously in other quadruplexes formed by minor-groove alignment of Watson-Crick base pairs. However, in this case the complexes are heterodimeric and are formed by two different tetrads (G:C:A:T and G:C:G:C). These complexes represent a new model of DNA recognition by small cyclic oligonucleotides, increasing the number of potential applications of these interesting molecules.