Ebbe Sloth Andersen

Self-assembly of a nanoscale DNA box with a controllable lid

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a ‘bottom-up’ approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA ‘origami’ method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 × 36 × 36 nm3 in size that can be opened in the presence of externally supplied DNA ‘keys’. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.

DNA Origami Design of Dolphin-Shaped Structures with Flexible Tails

The DNA origami method allows the folding of long, single-stranded DNA sequences into arbitrary two-dimensional structures by a set of designed oligonucleotides. The method has revealed an unexpected strength and efficiency for programmed self-assembly of molecular nanostructures and makes it possible to produce fully addressable nanostructures with wide-reaching application potential within the emerging area of nanoscience. Here we present a user-friendly software package for designing DNA origami structures ( http://www.cdna.dk/origami ) and demonstrate its use by the design of a dolphin-like DNA origami structure that was imaged by high-resolution AFM in liquid. The software package provides automatic generation of DNA origami structures, manual editing, interactive overviews, atomic models, tracks the design history, and has a fully extendable toolbox. From the AFM images, it was demonstrated that different designs of the dolphin tail region provided various levels of flexibility in a predictable fashion. Finally, we show that the addition of specific attachment sites promotes dimerization between two independently self-assembled dolphin structures, and that these interactions stabilize the flexible tail.

A single-stranded architecture for cotranscriptional folding of RNA nanostructures

The future of RNA origami writ large Researchers have long fabricated intricate nanostructures from carefully linked DNA strands. Now they can use RNA made by gene expression, which avoids the costly strand synthesis and lengthy annealing steps necessary with DNA origami. Geary et al. used molecular modeling to extend the size of folded RNA origami structures (see the Perspective by Leontis and Westhof). The modeling revealed assembly patterns for linking single-stranded RNA into A-form helices. The authors created two-dimensional structures as large as 660 nucleotides on mica surfaces. Science, this issue p. 799; see also p. 732 The size of RNA origami nanostructures has been increased with a distinct assembly pattern. [Also see Perspective by Leontis and Westhof] Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells. We introduce an architecture for designing artificial RNA structures that fold from a single strand, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern. We constructed RNA tiles that assemble into hexagonal lattices and demonstrated that lattices can be made by annealing and/or cotranscriptional folding. Tiles can be scaled up to 660 nucleotides in length, reaching a size comparable to that of large natural ribozymes.

In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: A cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12single), 20 as +12 with additional abnormalities (+12complex). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5+, Slg(weak), mainly FMC7-) and atypical for CLL in 122 patients (25%) (CD5-, or Slg(strong) or FMC7+). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) (P < 0.00005). Also +12complex, 14q+, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q+, an abnormal karyotype, +12complex, more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunophenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12complex, 14q+, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

The tmRDB and SRPDB resources.

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL with mirror sites located at Auburn University, Auburn, Alabama () and the Royal Veterinary and Agricultural University, Denmark (). The signal recognition particle database (SRPDB) at is mirrored at and the University of Goteborg (). The databases assist in investigations of the tmRNP (a ribonucleoprotein complex which liberates stalled bacterial ribosomes) and the SRP (a particle which recognizes signal sequences and directs secretory proteins to cell membranes). The curated tmRNA and SRP RNA alignments consider base pairs supported by comparative sequence analysis. Also shown are alignments of the tmRNA-associated proteins SmpB, ribosomal protein S1, alanyl-tRNA synthetase and Elongation Factor Tu, as well as the SRP proteins SRP9, SRP14, SRP19, SRP21, SRP54 (Ffh), SRP68, SRP72, cpSRP43, Flhf, SRP receptor (alpha) and SRP receptor (beta). All alignments can be easily examined using a new exploratory browser. The databases provide links to high-resolution structures and serve as depositories for structures obtained by molecular modeling.

Construction of a 4 Zeptoliters Switchable 3D DNA Box Origami

The DNA origami technique is a recently developed self-assembly method that allows construction of 3D objects at the nanoscale for various applications. In the current study we report the production of a 18 × 18 × 24 nm(3) hollow DNA box origami structure with a switchable lid. The structure was efficiently produced and characterized by atomic force microscopy, transmission electron microscopy, and Förster resonance energy transfer spectroscopy. The DNA box has a unique reclosing mechanism, which enables it to repeatedly open and close in response to a unique set of DNA keys. This DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing.

Role of the Trans-activation Response Element in Dimerization of HIV-1 RNA

The HIV-1 genome consists of two identical RNA strands that are linked together through non-covalent interactions. A major determinant for efficient dimerization of the two RNA strands is the interaction between palindromic sequences in the dimerization initiation site. Here we use an interplay of bioinformatics, biochemistry, and atomic force microscopy to describe another conserved palindrome in the trans-activation response element (TAR) that functions as a strong dimerization site when transiently exposed to the viral nucleocapsid protein. In conjunction with the DIS interaction, the TAR dimerization induces the formation of a 65-nm higher-order circular structure in the dimeric HIV-1 RNA. Our results provide a molecular model for the role of TAR in packaging and reverse transcription of the viral genome. The unique structure of the TAR-TAR dimer renders it an intriguing therapeutic target for the treatment of HIV-1 infection.

Dimerization and Template Switching in the 5′ Untranslated Region between Various Subtypes of Human Immunodeficiency Virus Type 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

Prediction and design of DNA and RNA structures.

Computational tools for prediction and design of DNA and RNA structures are used for different approaches in nucleic acid research. The prediction tools are used for the identification and modeling of biologically important nucleic acid structures, whereas the design tools aim at constructing novel molecular architectures and devices for nanotechnology and synthetic biology. The recent successes in predicting RNA three-dimensional structure directly from sequence and in designing sequences that self-assemble into predefined DNA and RNA nanostructures show that nucleic acid structure is predictable and controllable. The prediction and design approaches deal with reverse problems of relating sequence and structure, but share the main computational principles, visual representations and modeling approaches. The prediction and design tools are introduced together here to provide an overview of their current capabilities and deficiencies. The tools are listed by input and output to provide a user perspective and an extended tool table is made available at http://www.cdna.dk/tools/.

Multithreaded comparative RNA secondary structure prediction using stochastic context-free grammars

BackgroundThe prediction of the structure of large RNAs remains a particular challenge in bioinformatics, due to the computational complexity and low levels of accuracy of state-of-the-art algorithms. The pfold model couples a stochastic context-free grammar to phylogenetic analysis for a high accuracy in predictions, but the time complexity of the algorithm and underflow errors have prevented its use for long alignments. Here we present PPfold, a multithreaded version of pfold, which is capable of predicting the structure of large RNA alignments accurately on practical timescales.ResultsWe have distributed both the phylogenetic calculations and the inside-outside algorithm in PPfold, resulting in a significant reduction of runtime on multicore machines. We have addressed the floating-point underflow problems of pfold by implementing an extended-exponent datatype, enabling PPfold to be used for large-scale RNA structure predictions. We have also improved the user interface and portability: alongside standalone executable and Java source code of the program, PPfold is also available as a free plugin to the CLC Workbenches. We have evaluated the accuracy of PPfold using BRaliBase I tests, and demonstrated its practical use by predicting the secondary structure of an alignment of 24 complete HIV-1 genomes in 65 minutes on an 8-core machine and identifying several known structural elements in the prediction.ConclusionsPPfold is the first parallelized comparative RNA structure prediction algorithm to date. Based on the pfold model, PPfold is capable of fast, high-quality predictions of large RNA secondary structures, such as the genomes of RNA viruses or long genomic transcripts. The techniques used in the parallelization of this algorithm may be of general applicability to other bioinformatics algorithms.