RNA-guided DNA assembly

We propose molecular models for homologous DNA recombination events that are guided by either double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) templates. The models are applied to explain DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha, where extensive gene rearrangement occurs during differentiation of a somatic macronucleus from a germline micronucleus. We describe a model for RNA template guided DNA recombination, such that the template serves as a catalyst that remains unchanged after DNA recombination. This recombination can be seen as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a virtual knot diagram can provide a physical representation of the DNA at the time of recombination. Schematically, the braiding process can be represented as a crossing in the virtual knot diagram. The homologous recombination corresponds to removal of the crossings in the knot diagram (called smoothing). We show that if all recombinations are performed at the same time (i.e., simultaneous smoothings of the crossings) then one of the resulting DNA molecules will always contain all of the gene segments in their correct, linear order, which produces the mature DNA sequence.     

NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.     

Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.     

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.     

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.     

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.     

DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids.

Helicases not only catalyse the disruption of hydrogen bonding between complementary regions of nucleic acids, but also move along nucleic acid strands in a polar fashion. Here we show that the Rep52 and Rep40 proteins of adeno-associated virus type 2 (AAV-2) are required to translocate capsid-associated, single-stranded DNA genomes into preformed empty AAV-2 capsids, and that the DNA helicase function of Rep52/40 is essential for this process. Furthermore, DNase protection experiments suggest that insertion of AAV-2 genomes proceeds from the 3' end, which correlates with the 3'-->5' processivity demonstrated for the Rep52/40 helicase. A model is proposed in which capsid-immobilized helicase complexes act as molecular motors to 'pump' single-stranded DNA across the capsid boundary.     

Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.     

Thermodynamic contributions of the reactions of DNA intramolecular structures with their complementary strands

One focus of our research is to further our understanding of the physico-chemical properties of unusual DNA structures and their interaction with complementary oligonucleotides. We have investigated three types of reactions involving the interaction of intramolecular DNA complexes with their complementary single strands of varied length. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning (DSC) calorimetry and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of an i-motif, G-quadruplex, and triplex with their complementary strands. The enthalpies for each reaction are measured directly in ITC titrations and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA complex. These favorable free energy terms are enthalpy driven, which result from a compensation of exothermic contributions, due to the formation of additional base-pair stacks (or base-triplet stacks) in the duplex product (or triplex product), immobilization of electrostricted water by the base-pair and base-triplet stacks, and the removal of structural water from the reactant single strands; and endothermic contributions from the disruption of base-base stacking interactions of the reactant single strands. This investigation of nucleic acid reactions has provided new methodology, based on physico-chemical principles, to determine the molecular forces involved in the interactions between DNA nucleic acid structures. This methodology may be used in targeting reactions for the control of gene expression.     

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.     

Non-hexameric DNA helicases and translocases: mechanisms and regulation

Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.     

Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions

Simian virus 40 large T antigen is a helicase separating the complementary strands of double-stranded DNA in the presence of hydrolyzable ATP and of double-stranded RNA in the presence of non-ATP nucleotides (GTP, CTP or UTP). We have constructed partially single-stranded nucleic acid substrates consisting of RNA or DNA strands hydrogen bonded to either RNA or DNA complements. We found that ATP is utilized as a cofactor for the T-antigen-catalyzed unwinding reaction when the substrates contain overhanging single-stranded DNA, regardless of whether the double-stranded region is DNA or hybrid DNA.RNA. Conversely, non-ATP nucleotides are used when the overhanging single strand is RNA. Based on these and additional findings, we propose that the bound nucleic acid induces a conformational change in T antigen resulting in a proper orientation of both nucleic acid and nucleotide relative to the active center of the ATPase/helicase domain of the enzyme. The implications of our conclusion for the roles which T antigen may play in vivo are discussed.     

Complementary addressed modification of single- and double-stranded DNA by alkylating derivatives of oligonucleotides isolated by partial DNA fragmentation

Reagents for complementary addressed modification of nucleic acids are proposed to be synthesized on the base of oligonucleotides obtained by partial chemical fragmentation of DNA. The alkylating 4-(N-2 chlorethyl-N-methylamino) benzyl-5'-phosphamide derivatives of 5'-[32P]-labelled oligonucleotides obtained from single and double-stranded DNA cloned in bacteriophage M13 mp9 have been synthesized. The alkylated derivatives of oligonucleotides selectively modify the complementary tracts of single-stranded DNA-target. They are also able to modify the complementary regions in double-stranded supercoiled plasmid DNA.     

Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA

The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.     

Determining the influence of structure on hybridization using oligonucleotide arrays.

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.