Strand invasion of mixed-sequence, double-helical B-DNA by γ-peptide nucleic acids containing G-clamp nucleobases under physiological conditions

Peptide nucleic acids (PNAs) make up the only class of nucleic acid mimics developed to date that has been shown to be capable of invading double-helical B-form DNA. Recently, we showed that sequence limitation associated with PNA recognition can be relaxed by utilizing conformationally preorganized γ-peptide nucleic acids (γPNAs). However, like all the previous studies, with the exception of triplex binding, DNA strand invasion was performed at relatively low salt concentrations. When physiological ionic strengths were used, little to no binding was observed. On the basis of this finding, it was not clear whether the lack of binding is due to the lack of base pair opening or the lack of binding free energy, either of which would result in no productive binding. In this work, we show that it is the latter. Under simulated physiological conditions, the DNA double helix is sufficiently dynamic to permit strand invasion by the designer oligonucleotide molecules provided that the required binding free energy can be met. This finding has important implications for the design oligonucleotides for recognition of B-DNA via direct Watson-Crick base pairing.     

Triplex formation involving 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) with 1-isoquinolone base analogue: efficient and selective recognition of C:G interruption

For the effective recognition of C x G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O, 4'-C-methylene bridged nucleic acid with 1-isoquinolone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Triplex formation involving 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) with 2-pyridone base analogue: efficient and selective recognition of C:G interruption

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O,4'-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

Recent improvements in antigene technology

DNA triple-helix-based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother-like treatment in comparison to the flourishing field of oligonucleotide-based control of translation (antisense technology). This was mostly due to lack of affinity of triplex-forming oligonucleotides to their DNA target, to sequence restriction constraints imposed by the triple helical recognition motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, for example, the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid sugar unit, which greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence-specific genomic DNA manipulation is starting to become a useful tool in biotechnology.     

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 2-Pyridone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O,4′-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 1-Is oquinolone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C ? G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O, 4′-C-methylene bridged nucleic acid with 1-isoquinolone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

三链DNA稳定性的影响因素

介绍了碱基组成、碱基修饰或替代、DNA骨架的修饰、DNA配体的结合及反应体系中的盐离子和pH值等因素对三链DNA稳定性的影响。对三链DNA稳定性研究中应注意的几个问题也作了讨论。     

Recognition of double-stranded RNA by guanidine-modified peptide nucleic acids

Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Herein, we tested if the molecular recognition of RNA could be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from d-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.     

Searching genomes for sequences with the potential to form intrastrand triple helices

The canonical double-helix form of DNA is thought to predominate both in dilute solution and in living cells. Sequence-dependent fluctuations in local DNA shape occur within the double helix. Besides these relatively modest variations in shape, more extreme and remarkable structures have been detected in which some bases become unpaired. Examples include unusual three-stranded structures such as H-DNA. Certain RNA and DNA strands can also fold onto themselves to form intrastrand triplexes. Although they have been extensively studied in vitro, it remains unknown whether nucleic acid triplexes play natural roles in cells.If natural nucleic acid triplexes were identified in cells, much could be learned by examining the formation, stabilization, and function of such structures. With these goals in mind, we adapted a pattern-recognition program to search genetic databases for a type of potential triplex structure whose presence in genomes has not been previously investigated. We term these sequences Potential Intrastrand Triplex (PIT) elements. The formation of an intrastrand triplex requires three consecutive sequence domains with appropriate symmetry along a single nucleic acid strand. It is remarkable that we discovered multiple copies of sequence elements with the potential to form one particular class of intrastrand triplexes in the fully sequenced genomes of several bacteria. We then focused on the characterization of the 25 copies of a particular approximately 37 nt PIT sequence detected in Escherichia coli. Through biochemical studies, we demonstrate that an isolated DNA strand from this family of E. coli PIT elements forms a stable intrastrand triplex at physiological temperature and pH in the presence of physiological concentrations of Mg(2+).     

Recognition of duplex DNA by RNA polynucleotides.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence.     

肽核酸在分子生物学技术中的应用

肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同。PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构。与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点。简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望。     

Determination of the optimized conditions for coupling oligonucleotides with 16-mercaptohexadecanoic acid chemically adsorbed upon Au

A specific 5'-modified amino group oligonucleotide (Primer 1), 15-mers in length, is selectively coupled with the carboxyl terminated 16-mercaptohexadecanoic acid (MHDA) chemically adsorbed on Au and subsequently hybridized with Antisense Primer. The amide-coupling process is of significance to create an intermediate structure for the purpose of adding Primer 1, while the hybridization reaction is relevant to various diagnostic purposes to determine the presence in nucleic acids for a target sequence. In this work, the coupling setting was particularly emphasized by varying commonly used temperatures and pH values with a definite concentration of coupling agents (i.e., 10 mM). The recombination with analogous hybridization treatment was investigated using high resolution X-ray photoelectron spectroscopy and a 75 degrees grazing angle Fourier transform infrared spectrometer. On the basis of the spectroscopic studies, the optimized conditions for the coupling process that is also correlated with the molecular density of subsequent hybridization process on MHDA/Au have been proposed at 37 degrees C and a pH value of 4.5. Therefore, it is pertinent to intensify the joining of short-chain DNA strands by complementary base pairing in diagnostic applications such as the identification of single nucleotide polymorphisms.     

Two sides of the coin: affinity and specificity of nucleic acid interactions

During the past decade, synthetic nucleobase oligomers have found wide use in biochemical sciences, biotechnology and molecular medicine, both as research and/or diagnostic tools and as therapeutics. Numerous applications of common and modified oligonucleotides and oligonucleotide mimics rely on their ability to sequence-specifically recognize nucleic acid targets (DNA or RNA) by forming duplexes or triplexes. In general, these applications would benefit significantly from enhanced binding affinities of nucleobase oligomers in the formation of various secondary structures. However, for high-affinity probes, the selectivity of sequence recognition must also be improved to avoid undesirable associations with mismatched DNA and RNA sites. Here, we review recent progress in understanding the molecular mechanisms of nucleic acid interactions and the development of new high-affinity plus high-specificity oligonucleotides and their mimics, with particular emphasis on peptide nucleic acids.     

Molecular beacons: nucleic acid hybridization and emerging applications.

Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.     

Peptide nucleic acids: deoxyribonucleic acid mimics with a peptide backbone

This tutorial briefly describes a new class of synthetic biopolymer, which is referred to as peptide nucleic acid (PNA). In PNA, individual nucleobases are linked to an achiral neutral peptide backbone. PNA exhibits the hybridization characteristic (e.g., Watson—Crick duplex formation) of DNA. The achiral peptide backbone provides similar interbase distances as natural DNA, and adequate flexibility to permit base pair interactions with complementary RNA or DNA strands. Several potential applications of PNA oligomers in biotechnology are suggested. These include the use of PNAs as a probe for specific recognition of a DNA or RNA sequence selective, purification of nucleic acids via designed high affinity binding to PNA, screening for DNA mutations, and as possible therapeutic agents.     

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.     

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.