The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.     

Synthesis and spectroscopic characterization of binaphthol aminosugars for stimulation of DNA strand slippage synthesis

Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Two new binaphthol aminosugars were first synthesized as DNA bulge binders to study the triplet repeat expansion due to the wedge-shaped structure of 1,1′-bi-2-naphthol. Both compounds were structurally characterized by 1- and 2-D NMR. They showed remarkable fluorescence enhancement when binding with bulge DNA and they exhibited stimulation for ATT·AAT trinucleotide repeat DNA sequence slippage synthesis.     

Highly fluorescence quenching graphene oxide-based oligodeoxynucleotide hairpin systems for probing CNG DNA repeat sequences

In this study we synthesized a novel graphene-oxide (GO) based CNG repeat hairpin probing system capable of detecting target CAG and CTG DNA repeat sequences. The fluorescence of the 30-mer CNG repeat hairpin structure was quenched dramatically by GO in the absence of the target sequence, with a high quenching constant [K = 0.030 (mg/mL)^?1]. We optimized the quenching behavior of this probing system by using graphene oxide (GO) to induce a high degree of discrimination factor (44.6 times) between the fluorescence of the target sequence and that of other non-target sequences. All detection process is explained by displacement mechanism using adsorption, desorption, and hybridization of probe with target DNA sequence on the GO. Graphene-oxide (GO) based CNG repeat hairpin probing system exhibited high sensitivity and selectivity to the target CNG repeat sequence and its detection process is so simple and quick.     

三螺旋脱氧核糖核酸的研究进展

阐述了三螺旋DNA的发展和最新动态,并从3个方面展开评述：(1)三螺旋DNA稳定性的研究。三螺旋DNA的稳定性不仅取决于寡聚核苷酸的内在结构,还受外界环境如溶液的pH值、阳离子的种类和价态、DNA分子嵌入试剂和共聚物等的影响;(2)三螺旋DNA的应用。三螺旋DNA的形成为基因操纵和基因疗法提供了新方法,它在抑制DNA转录、复制、基因定点诱变、定点切割和诱导基因重组等方面有重要的应用前景;(3)三螺旋DNA的检测方法,包括紫外-可见吸收光谱法、荧光分析法、原子力显微术和顺磁共振谱等。     

Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching

Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5'CUG/3'GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca(2+) ATPase 1, and cardiac troponin T. Based on these observations, the development of small-molecule ligands that target specifically expanded DM1 repeats could be of use as therapeutics. In the present study, chemical similarity searching was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of in vitro inhibitors of the RNA-protein complex were identified with low micromolar IC(50)'s, which are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with chemical similarity searching.     

Detection of single and multiple polymorphic loci by synthetic tandem repeats of short oligonucleotides

Loci containing tandem repeats of short sequences are sometimes associated with a high level of polymorphism due to variations in the number of repeats. The different variants can be easily characterized by Southern blotting when the repeats span a range from a few hundred bases to a few kilobases, and probes derived from such tandem repeats constitute convenient genetic markers. These structures, usually called minisatellites, are best documented in the human genome, where their number has been estimated to be at least 1500. However, their role and mode of evolution are poorly understood. We are developing tools to evaluate the number of such redundant sequences in a genome and to gain access to new polymorphic loci. Our strategy is based on the use of polymers of oligonucleotides as DNA probes for hybridization on Southern blots. In a previous report, we made polymers with random units of 14 bp and showed that they detect multiple polymorphic loci on human genomic DNA. At present, we are testing the effect of an increase in the complexity of the polymer, as obtained by the use of a longer random unit, and the effect of slight sequence modifications to a particular tandem repeat sequence. In addition, some of these synthetic probes can detect a single polymorphic locus and directly provide new genetic markers.     

Naphthyridine‐Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA

The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small‐molecule naphthyridine‐azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA ‐binding to the CAG repeat DNA and RNA, we conducted systematic structure‐binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA , modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)₉ and r(CAG)₉. NBzA binding to d(CAG)₉ was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry. For the binding to r(CAG)₉, both NA and NBzA showed stepwise binding in ESI‐TOF MS, and NBzA ‐binding to r(CAG)₉ induced more extensive conformational change than NA ‐binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.     

Analytical validation of quality control materials for Huntington’s disease examination

The expansion of molecular diagnostics using nucleic acid technologies in clinical and public health practice has increased the need for appropriate reference materials and verified quality-control materials for quality assurance, test validation, proficiency testing and the development of new examination procedures. Good laboratory practice requires the use of reference materials to establish an examination procedure and assess the variability of the results. Reference materials are also required to assess the assay on a daily basis and to normalize results collected among different laboratories. Despite the growing volume, the rapidly increasing number of tests being offered, and the necessary routine use, certified reference materials are often not available. There is a wide range of human genetic bio-assays for which there are no available traceable certified reference materials. Several initiatives have been organized to provide well-characterized quality control specimens (e.g., cell lines) with known DNA mutations for use in diagnostics. Mutations are confirmed with bi-directional DNA sequence analysis, which is considered the reference examination procedure. In the field of personalized medicine, NIST has created and validated Standard Reference Material^® 2399 for fragile X examinations. Herein we describe our characterization of candidate reference materials for Huntington’s disease genetic examination. Bi-directional DNA sequencing confirmed the size of the CAG repeat contained on each allele from patient derived materials. Amplification and capillary electrophoresis of the CAG repeats had an uncertainty ranging from 2.06%CV to 7.83%CV.     

Triptycene-based small molecules modulate (CAG)·(CTG) repeat junctions

Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)·(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism.     

The frequency of poly(G) tracts in the human genome and their use as a sensor of DNA damage

Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n = 9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n > 9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences.     

Comparative binding of antitumor drugs to DNA containing the telomere repeat sequence

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 micromM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.     

Electrochemical detection of DNA triplet repeat expansion

Hereditary neurodegenerative diseases are connected with the expansion of trinucleotide repetitive sequences in genomic DNA. Molecular diagnosis of these diseases is based on the determination of the triplet repeat length. Currently used methods involve PCR amplification followed by electrophoretic determination of the amplicon size. We propose a novel electrochemical technique based on hybridization of target DNA (tDNA) immobilized at magnetic beads with a reporter probe (RP) complementary to the triplet repeats (12 units per RP). The biotin-labeled RP is detected via an enzyme-linked electrochemical assay involving binding of streptavidin-alkaline phosphatase conjugate and transformation of electroinactive 1-naphthyl phosphate to electroactive 1-naphthol. Pyrimidine residues within sequences flanking the homopurine (GAA)n repeat in tDNA are premodified with osmium tetroxide, 2,2'-bipyridine (Os,bipy), introducing electroactive labels in tDNA. The length of the triplet expansion is calculated from the ratio of the intensities of electrochemical signals of hybridized RP/tDNA-Os,bipy. The normalized signal increases linearly with the repeat length between 0 and about 200 triplet units, allowing for discrimination between normal, premutated, and mutated alleles. Application of this method for the detection of the asymptomatic heterozygous carrier of expanded alleles is demonstrated.     

Site-specific probing of oxidative reactivity and telomerase function using 7,8-dihydro-8-oxoguanine in telomeric DNA

Telomeres at the ends of human chromosomes contain the repeating sequence 5'-d[(TTAGGG)(n)]-3'. Oxidative damage of guanine in DNAs that contain telomeric and nontelomeric sequence generates 7,8-dihydro-8-oxoguanine (8OG) preferentially in the telomeric segment, because GGG sequences are more reactive in duplex DNA. We have developed a general strategy for probing site-specific oxidation reactivity in diverse biological structures through substitution of minimally modified building blocks that are more reactive than the parent residue, but preserve the parent structure. In this study, 8OG was substituted for guanine at G(8), G(9), G(14), or G(15) in the human telomeric oligonucleotide 5'-d[AGGGTTAG(8)G(9)GTT AG(14)G(15)GTTAGGGTGT]-3'. Replacement of G by 8OG in telomeric DNA can affect the formation of intramolecular G quadruplexes, depending on the position of substitution. When 8OG was incorporated in the 5'-position of a GGG triplet, G quadruplex formation was observed; however, substitution of 8OG in the middle of a GGG triplet produced multiple structures. A clear correspondence between structure and reactivity was observed when oligonucleotides containing 8OG in the 5'-position of a GGG triplet were prepared in the quadruplex or duplex forms and interrogated by mediated electrocatalytic oxidation with Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine). The rate constant for one-electron oxidation of a single 8OG in the 5'-position of a GGG triplet was (6.2 +/- 1.7) x 10(4) M(-1) s(-1) in the G quadruplex form. The rate constant was 2-fold lower for the same telomeric sequence in the duplex form ((3.0 +/- 1.3) x 10(4) M(-1) s(-1)). The position of 8OG in the GGG triplet affects telomerase activity and synthesis of telomeric repeat products. Telomerase activity was decreased significantly when 8OG was substituted in the 5'-position of the GGG triplet, but not when 8OG was substituted in the middle of the triplet. Thus, biological oxidation of G to 8OG in telomeres has the potential to modulate telomerase activity. Further, small molecules that inhibit telomerase by stabilizing telomeric G quadruplexes may not be as effective under oxidative stress.     

A Single‐Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double‐surface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single‐surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen‐printed) electrode. Biotin‐labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin‐alkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1‐naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme‐linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2′‐bipyridine complex. Detection of (GAA)_n?(TTC)_n triplet repeat expansion in nanogram quantities of PCR‐amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.     

Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

The reliable genetic diagnostics of triplet repeat expansions by capillary electrophoresis (CE) remains technically challenging due to the properties of the repeated GC-rich sequences. The biased base composition of the analyzed sample as compared to the commonly used DNA size standards makes the precise repeat length determination questionable. The homologous allelic ladders improve the accuracy of the repeat length analysis significantly. Their use, however, is not devoid of other complications. In the approach we propose, the allelic ladders are used only to properly calibrate the commercially available standards which serve then as internal standards in reliable and economical repeat length determination. In this study, we have also analyzed factors that could possibly increase the sensitivity of mutant allele detection by increasing the overall amplification efficiency and long-to-short product ratio.     

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.     

人类Y染色体回文序列中的重复序列与碱基关联

利用信息论和统计学的方法并结合生物学的特征研究人类Y染色体回文序列的互信息、“n字”熵、条件熵,定量分析了回文序列的长程关联和短程关联,发现其中既存在长程关联也存在短程关联,并且它们主要是由序列中的重复序列引起的．研究表明重复序列含量越高碱基之间的关联越强．