3D Structure Of DNA Research Articles

100 Bridging the gap: from 1D to 3D genomics

Combination of the nucleotides A, C, G, and T leads to one-dimensional (1D) DNA sequence that further gives rise to the three-dimensional (3D) structure of DNA. In fact, proteins do not literally read the letters A, C, G, and T. Instead, read out is based on spatial interactions between 3D objects, in which the structures of both DNA and proteins are important for the interactions to occur (Rohs et al. 2009; Rohs et al. 2010). On the other hand, while the 3D structure of DNA is dependent on the primary sequence, there is degeneracy in mapping DNA sequence to shape. Moreover, while high-throughput sequencing technologies continue to produce large amounts of DNA sequence information, experimental data on 3D structures of DNA is limited. These facts all urge to develop new approaches that provide new insights into DNA structure. Given such motivation, we have developed a high-throughput tool for predicting shape of “naked” DNA on a genomic scale (Slattery et al. 2011) and a web server for predicting DNA shape information. In parallel, we constructed a database of shape that features of transcription factor binding sites using sequence-based databases as the source for binding motifs. These tools provide a novel approach for studying transcription factor binding sites, especially those of paralogous transcription factors which share highly similar core-binding motifs but bind different genome sites in vivo. Our approach can also be used to study less-specific protein–DNA interactions, for example, histone–DNA interactions in nucleosomes. Here, we present structural profiles of transcription factor and nucleosome binding sites of various organisms and provide evidence that DNA shape generally plays an important role in protein–DNA recognition. Specifically, we present insights in how paralogous transcription factors recognize DNA shape to achieve differential binding specificity and suggest that mechanisms for nucleosome formation are somewhat different in Plasmodium falciparum whose genome has an extremely high A/T content compared with Saccharomyces cerevisiae and Drosophila melanogaster.

Zipper-Based Devices and Future Directions

A strand displacement-based system has been developed that substitutes inosine for guanosine in DNA sequences called “zippers”. Each zipper sequence employs traditional adenosine-thymine bonding as well as non-traditional inosine-cytidine bonding. The I-C bond consists of only 2 hydrogen bonds as opposed to the typical 3 hydrogen bonds found in G-C bonds. A zipper helix consists of one strand with A and C and a second strand with complementary I and T nucleotides. The second strand is displaced by the introduction of a strand with G and T nucleotides complementary to the first strand. These zippers can be introduced as an active element in larger DNA devices and be designed to be used in different situations. The first example is of zippers incorporated into single and double “spring” systems. The springs incorporate in such a manner that allow them be repeatedly extended and reset. Multiple springs can be incorporated into larger 2D and 3D DNA structures, allowing them to change between conformations on demand. Zippers can also be incorporated into a gating system for ion channels such as hemolysin and DNA tethered to a larger nanoparticle and passing through the hemolysin channel. Zippers at the end of such a strand could lock the DNA strand in place, effectively allowing the nanoparticle to block the channel on the other side. Finally DNA zippers have not been properly characterized by quantitative measurements yet. AFM or optical trap studies can be performed to study the binding energy of a zipper sequence.

Polarization-Sensitive Two-Photon Microscopy Study of the Organization of Liquid-Crystalline DNA

Highly concentrated DNA solutions exhibit self-ordering properties such as the generation of liquid-crystalline phases. Such organized domains may play an important role in the global chromatin topology but can also be used as a simple model for the study of more complex 3D DNA structures. In this work, using polarized two-photon fluorescence microscopy, we report on the orientation of DNA molecules in liquid-crystalline phases. For this purpose, we analyze the signal emitted by fluorophores that are noncovalently bound to DNA strands. In nonlinear processes, excitation occurs exclusively in the focal volume, which offers advantages such as the reduction of photobleaching of out-of-focus molecules and intrinsic 3D sectioning capability. Propidium iodide and Hoechst, two fluorophores with different DNA binding modes, have been considered. Polarimetric measurements show that the dyes follow the alignment with respect to the DNA strands and allow the determination of the angles between the emission dipoles and the longitudinal axis of the DNA double strand. These results provide a useful starting point toward the application of two-photon polarimetry techniques to determine the local orientation of condensed DNA in physiological conditions.

Assembly and structural analysis of a covalently closed nano-scale DNA cage.

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

A 3D pattern matching algorithm for DNA sequences

Biologists usually work with textual DNA sequences (succession of A, C, G and T). This representation allows biologists to study the syntax and other linguistic properties of DNA sequences. Nevertheless, such a linear coding offers only a local and a one-dimensional vision of the molecule. The 3D structure of DNA is known to be very important in many essential biological mechanisms. By using 3D conformation models, one is able to construct a 3D trajectory of a naked DNA molecule. From the various studies that we performed, it turned out that two very different textual DNA sequences could have similar 3D structures. In this article, we address a new research work on 3D pattern matching for DNA sequences. The aim of this work is to enhance conventional pattern matching analyses with 3D-augmented criteria. We have developed an algorithm, based on 3D trajectories, which compares angles formed by these trajectories and thus quantifies the difference between two 3D DNA sequences. This analysis performs from a global scale to al local one. Available on request from the authors.

1P012 The 3D structure of DNA polymerase/PCNA/DNA complex obtained from single particle EM images(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P012 The 3D structure of DNA polymerase/PCNA/DNA complex obtained from single particle EM images(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Synthesis and conformational properties of oligonucleotides incorporating 2'-O-phosphorylated ribonucleotides as structural motifs of pre-tRNA splicing intermediates.

To synthesize oligonucleotides containing 2'-O-phosphate groups, four kinds of ribonucleoside 3'-phosphoramidite building blocks 6a-d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) group were prepared according to our previous phosphorylation procedure. These phosphoramidite units 6a-d were not contaminated with 3'-regioisomers and were successfully applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22. Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2'-O-phosphorylated ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out that KI(3) was the most effective reagent for oxidative desulfurization of the initially generated thiophosphate group to the phosphate group on polymer supports. Without using this conversion step, a tridecadeoxyuridylate 17 incorporating a 2'-O-thiophosphorylated uridine derivative was also synthesized. To investigate the effect of the 2'-phosphate group on the thermal stability and 3D-structure of DNA(RNA) duplexes, T(m) measurement of the self-complementary oligonucleotides obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2'-hydroxyl function predominantly preferred C2'-endo to C3'-endo conformation in DNA duplexes so that it did not significantly affect the stability of the DNA duplex. On the other hand, the 2'-modified ribose moiety was expelled to give a C3'-endo conformation in RNA duplexes so that the RNA duplexes were extremely destabilized.