Double-helical DNA Sequences Research Articles

Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes.

We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator-like effector proteins folds a double-stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation.

Genetically Encoded DNA-Protein Hybrid Origami

Here we describe an approach to bottom-up fabrication with nanometer-precision that allows integrating the functional diversity of proteins in designed three-dimensional structural frameworks. We reimagined the successful DNA origami design principle using a set of custom staple proteins to fold a double-stranded DNA template into a user-defined shape. Each staple protein recognizes two distinct double-helical DNA sequences and can carry additional functionalities. The staple proteins we present here are based on the transcription activator-like (TAL) effector proteins. Due to their repetitive structure these proteins offer a unique programmability that enables us to construct numerous staple proteins targeting any desired DNA sequence. Our approach is general, meaning that many different objects may be created using the same set of rules, and it is modular, because components can be modified or exchanged individually. We present rules for constructing megadalton-scale DNA-protein hybrid nanostructures; introduce important structural motifs, such as curvature, corners, and vertices; describe principles for creating multi-layer DNA-protein objects with enhanced rigidity; and demonstrate the possibility to combine our DNA-protein hybrid origami with conventional DNA nanotechnology. Since all components can be encoded genetically, our structures should be amenable to biotechnological mass-production. Moreover, since the target objects can self-assemble at room temperature in near-physiological buffer, our hybrid origami may also provide an attractive method to realize positioning and scaffolding tasks in vivo. We expect our method to find application both in scaffolding protein functionalities and in manipulating the spatial arrangement of genomic DNA.

Minor-Groove Binding Drugs: Where Is the Second Hoechst 33258 Molecule?

Hoechst 33258 binds with high affinity into the minor groove of AT-rich sequences of double-helical DNA. Despite extensive studies of this and analogous DNA binding molecules, there still remains uncertainty concerning the interactions when multiple ligand molecules are accommodated within close distance. Albeit not of direct concern for most biomedical applications, which are at low drug concentrations, interaction studies for higher drug binding are important as they can give fundamental insight into binding mechanisms and specificity, including drug self-stacking interactions that can provide base-sequence specificity. Using circular dichroism (CD), isothermal titration calorimetry (ITC), and proton nuclear magnetic resonance ((1)H NMR), we examine the binding of Hoechst 33258 to three oligonucleotide duplexes containing AT regions of different lengths: [d(CGCGAATTCGCG)]2 (A2T2), [d(CGCAAATTTGCG)]2 (A3T3), and [d(CGAAAATTTTCG)]2 (A4T4). We find similar binding geometries in the minor groove for all oligonucleotides when the ligand-to-duplex ratio is less than 1:1. At higher ratios, a second ligand can be accommodated in the minor groove of A4T4 but not A2T2 or A3T3. We conclude that the binding of the second Hoechst to A4T4 is not cooperative and that the molecules are sitting with a small separation apart, one after the other, and not in a sandwich structure as previously proposed.

Interaction Studies of an Anticancer Alkaloid, (+)-(13aS)-Deoxytylophorinine, with Calf Thymus DNA and Four Repeated Double-Helical DNAs

Background: Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity. The specific biomolecular targets of these alkaloids have not yet been clearly identified. (+)-(13aS)-deoxytylophorinine is a new phenanthroindolizidine alkaloid originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata in our institute. (+)-(13aS)-deoxytylophorinine exerts both in vitro and in vivoanticancer activities. Methods: The in vivo anticancer effects and toxicity of this compound were investigated in mice, and interactions between this compound and double-helical DNA sequences were studied in detail with circular dichroic spectroscopy and fluorescence spectroscopy. Viscosity measurements were applied to check the interactive mode between this compound and DNA. Results: Potent anticancer effects were observed in vivo. Also, concentration-dependent interactions were observed and this compound seemed to interact in a sequence-specific manner with AT-repeated sequences of double-helical DNA. Such interactions were proved to be intercalating by viscosity measurements. Conclusions: Anticancer alkaloid (+)-(13aS)-deoxytylophorinine can have sequence-specific interactions with DNA in an intercalating manner.

Triplex-Directed Interstrand DNA Cross-Linking by Diaziridinylquinone−Oligonucleotide Conjugates

Triplex-forming oligonucleotides (TFOs) bind in the major groove to specific double-helical DNA sequences and have been shown to inhibit the function of targeted genes. Diaziridinylquinones are bifunctional alkylating agents that can form interstrand cross-links in DNA at 5‘-GNC sites. To demonstrate the feasibility of targeted interstrand cross-linking, a diaziridinylquinone−TFO conjugate was prepared from a diaziridinylquinone intermediate bearing an activated ester linker. The first demonstration of triplex-directed interstrand DNA cross-linking by a single targeted bifunctional alkylating agent is described. Up to 38% interstrand cross-linking was observed at pH 6.2.

Triple helices formed at oligopyrimidine*oligopurine sequences with base pair inversions: effect of a triplex-specific ligand on stability and selectivity.

Oligonucleotide-directed triple helix formation is mostly restricted to oligopyrimidine*oligopurine sequences of double helical DNA. An interruption of one or two pyrimidines in the oligopurine target strand leads to a strong triplex destabilisation. We have investigated the effect of nucleotide analogues introduced in the third strand at the site opposite the base pair inversion(s). We show that a 3-nitropyrrole derivative (M) discriminates G*C from C*G, A*T and T*A in the presence of a triplex-specific ligand (a benzo[e]pyridoindole derivative, BePI). N6-methoxy-2,6-diaminopurine (K) binds to an A*T base pair better than a T*A, G*C or C*G base pair. Some discrimination is still observed in the presence of BePI and triplex stability is markedly increased. These findings should help in designing BePI-oligonucleotide conjugates to extend the range of DNA sequences available for triplex formation.

Synthesis of 13H-Benzo[6,7]- and 13H-Benzo[4,5]indolo[3,2-c]- quinolines: A New Series of Potent Specific Ligands for Triplex DNA

Triple-helical complexes formed upon binding of oligonucleotides to oligopyrimidine·oligopurine sequences of double-helical DNA can be stabilized by intercalating ligands such as benzopyridoindole derivatives (Mergny et al. Science 1992, 256, 1681). Based on molecular modeling studies, it was predicted that better stacking interactions could be achieved between the intercalator and base triplets by extending the size of the aromatic ring system. Here we describe the synthesis of pentacyclic aromatic molecules which exhibit a highly selective binding to triplex structures. The thermal Fischer indolization of hydrazones resulting from 4-hydrazinoquinolin-2(1H)-one and 6-methoxy-1 (and -2) -tetralones led to the expected cyclized intermediates. After complete aromatization, these compounds were transformed by phosphorus oxychloride giving 6-chloro-10-methoxy-13H-benzo[6,7]- (and 6-chloro-9-methoxy-13H-benzo[4,5]-) indolo[3,2-c]quinolines. Usual substitution by various diamines provided derivatives of these p...

Recognition of alternating oligopurine/oligopyrimidine tracts of DNA by oligonucleotides with base-to-base linkages.

A new concept is presented to design and synthesize modified oligonucleotides in order to extend the range of double-helical DNA sequences that can be recognized by oligonucleotides via triple helix formation. The DNA target is composed of adjacent oligopurine.oligopyrimidine domains where the oligopurine sequences alternate on the two DNA strands. Canonical (C,T)-motif triple helices are formed with each oligopurine.oligopyrimidine domain of the target sequence. The two third-strand oligonucleotides were joined together via an appropriate linker between the two terminal bases with either a 3'-3' or a 5'-5' polarity. Molecular modeling was used to predict the optimal length of the linker bridging two terminal bases. The interaction of DNA with such a modified oligonucleotide containing a C3'-3'U linkage was studied by thermal dissociation, footprinting, and gel retardation experiments. They provide experimental evidence that the oligonucleotide does form a switched triple helix on this extended DNA target sequence. The binding of the so-called "switch oligonucleotide" is enhanced as compared to the two unlinked parental oligonucleotides which form triple helices with each oligopurine.oligopyrimidine domain of the target sequence.

Rational design of sequence-specific DNA ligands for artificial control of gene expression

Abstract

DNA triple-helix formation: an approach to artificial gene repressors?

Certain sequences of double-helical DNA can be recognized and tightly bound by oligonucleotides. The effects of such triple-helical structures on DNA binding proteins have been studied. Stabilities of DNA triple-helices at or near physiological conditions are sufficient to inhibit DNA binding proteins directed to overlapping sites. Such proteins include restriction endonucleases, methylases, transcription factors, and RNA polymerases. These and other results suggest that oligonucleotide-directed triple-helix formation could provide the basis for designing artificial gene repressors. The general question of whether biological systems employ RNA molecules for recognition and regulation of double-helical DNA is discussed.

Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving.

Nearest neighbor interactions affect the stabilities of triple-helical complexes. Within a pyrimidine triple-helical motif, the relative stabilities of natural base triplets T.AT, C + GC, and G.TA, as well as triplets, D3.TA and D3.CG, containing the nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3) were characterized by the affinity cleaving method in the context of different flanking triplets (T.AT, T.AT: T.AT, C + GC: C + GC, T.AT: G + GC, C + GC). The to be insensitive to substitutions in either the 3' or 5' directions, while the relative stabilities of triple helices containing C + GC triplets decreased as the number of adjacent C + GC triplets increased. Triple helices incorporating a G.TA interaction were most stable when this triplet was flanked by two T.AT triplets and were adversely affected when a C + GC triplet was placed in the adjacent 5' direction. Similarly, complexes containing D3.TA or D3.CG triplets were most stable when the triplet was flanked by two T.AT triplets but were destabilized when the adjacent 3' neighbor position was occupied with a C + GC triplet. This information regarding sequence composition effects in triple-helix formation establishes a set of guidelines for targeting sequences of double-helical DNA by the pyrimidine triple-helix motif.

Mechanism of Sequence-Specific DNA Recognition Elucidated

In the past decade, researchers have begun investigations into the chemistry of such molecular interactions. One of those researchers is Peter B. Dervan, a chemistry professor at Calfiornia Institute of Technology in Pasasdena. Late last year, Dervan and Caltech coworkers published four papers that represent, in a sense, the culmination of about seven years of extremely successful research into the question of how molecules recognize and bind to specific sequences of double helical DNA. This article discusses that research.

A synthetic peptide binds 16 base pairs of A,T double helical DNA

One approach to the design of synthetic sequence specific DNA binding molecules that bind large sequences of double helical DNA is to couple together DNA binding domains of similar or diverse base pairs specificities. The DNA binding domains should be linked together in a way that allows simultaneous binding of the units to contiguous DNA sequences.

Proposed α-helical super-secondary structure associated with protein-DNA recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.