Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces.

Rich's biggest discoveries were left-handed Z-DNA, the tertiary structure of tRNA, and that DNA and RNA strands could pair up. His discoveries provided the groundwork for our understanding of nucleic acid structure and consequently the development of the fields of molecular biology, biotechnology, and nanotechnology. Rich's biggest discoveries were left-handed Z-DNA, the tertiary structure of tRNA, and that DNA and RNA strands could pair up. His discoveries provided the groundwork for our understanding of nucleic acid structure and consequently the development of the fields of molecular biology, biotechnology, and nanotechnology. Alexander Rich died on 27 April 2015 at the age of 90. Many obituaries were written about him by his friends and colleagues to testify to his enormous and unique overarching status as an academic teacher, scientist, colleague, and human companion. The aim of this retrospective is to recall his sometimes forgotten seminal contributions that laid the groundwork for modern biotechnology and in particular diagnostics, forensics, genealogy, genome sequencing, and many of the methods and tools used to detect genes in tissues. For the past 60 years, Rich deeply investigated nucleic acids. His interest in their structure was inspired by the double-helical model of DNA proposed by Watson and Crick [1Watson J.D. Crick F.H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (8503) Google Scholar]. Rich focused on the structure of RNA and was the RNA Tie Club member named for arginine (https://en.wikipedia.org/wiki/RNA_Tie_Club). When the double-helical DNA model was first proposed, there was doubt that RNA could also form such a double-helical structure due to van der Waals interference of the 2′-OH in ribose. However, in 1956 Rich found that mixing together polyriboadenylic acid [poly (A)] and polyuridine acid [poly (U)] was associated with a drop in optical density in UV light. This was the first demonstration that RNA molecules can form a double helix (molecular sex), which contributed to today's knowledge of nucleic acid structures [2Rich A. Davies D.R. A new two-stranded helical structure: polyadenylic acid and polyuridylic acid.J. Am. Chem. Soc. 1956; 78: 3548-3549Crossref Scopus (55) Google Scholar]. In 1957 he discovered that the poly (A) • poly (U) duplex could take on a third strand of poly (U) to form a triple helix [3Felsenfeld G. Rich A. Studies on the formation of two- and three-stranded polyribonucleotides.Biochim. Biophys. Acta. 1957; 26: 457-468Crossref PubMed Scopus (231) Google Scholar]. Evidence for triple-helical complexes of RNA molecules made it reasonable to ask whether DNA and RNA could interact and to consider a model in which RNA molecules were assembled by binding in a sequence-specific manner to double-stranded DNA. This proved to be the case and represented the first experimental demonstration that a hybrid helix could be a method for the transcription of information from DNA to RNA [4Rich A. A hybrid helix containing both deoxyribose and ribose polynucleotides and its relation to the transfer of information between the nucleic acids.Proc. Natl. Acad. Sci. U.S.A. 1960; 46: 1044-1053Crossref PubMed Google Scholar]. Notably, this was 1 year earlier than the discovery of mRNA in 1961 [5Brenner S. et al.An unstable intermediate carrying information from genes to ribosomes for protein synthesis.Nature. 1961; 190: 576-581Crossref PubMed Scopus (386) Google Scholar]. Rich's work on RNA structure led to his visionary hypothesis that life on Earth evolved from an ‘RNA World’ [6Rich A. On the problems of evolution and biochemical information transfer.in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 1962: 103-126Google Scholar]. He postulated that primitive polynucleotide chains were able to act as a template or as a somewhat inefficient catalyst for promoting the polymerization of complementary nucleotide residues to produce a double-stranded molecule [6Rich A. On the problems of evolution and biochemical information transfer.in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 1962: 103-126Google Scholar]. Additionally, Rich was the first to mention antisense RNA, in which two complementary RNA strands made inside the cell would hybridize [6Rich A. On the problems of evolution and biochemical information transfer.in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 1962: 103-126Google Scholar]. The significance of these ideas was not obvious at the beginning of the 1960s and was not fully recognized until years later. His work also included characterizing aspects of translation. Rich contributed to the discovery that multiple ribosomes are organized on mRNA at the same time. He observed that the efficient and rapid synthesis of polypeptides by ‘polyribosomes’ somewhat resembles train cars (ribosomes) on a track (mRNA) [7Warner J.R. et al.A multiple ribosomal structure in protein synthesis.Proc. Natl. Acad. Sci. U.S.A. 1963; 49: 122-129Crossref PubMed Scopus (244) Google Scholar]. In 1968, 10 years after the discovery tRNA, Rich's pioneering work produced the first crystals of bacterial initiator tRNAMet [8Kim S.H. Rich A. Single crystals of transfer RNA: an X-ray diffraction study.Science. 1968; 162: 1381-1384Crossref PubMed Scopus (47) Google Scholar]. Then, in 1973, researchers led by Rich solved the structure of tRNAPhe at 4-Å resolution. The folded tRNA chain identified the anticodon loop and CCA acceptor end and Watson–Crick base pairing in double-stranded stems as well as many noncanonical base pairs [9Kim S.H. et al.Three-dimensional structure of yeast phenylalanine transfer RNA: folding of the polynucleotide chain.Science. 1973; 179: 285-288Crossref PubMed Scopus (288) Google Scholar]. In addition, his laboratory described the well-known L-shaped model of tRNA structure at a resolution of 3 Å in 1974 (Figure 1) [10Suddath F.L. et al.Three-dimensional structure of yeast phenylalanine transfer RNA at 3.0 Ångstroms resolution.Nature. 1974; 248: 20-24Crossref PubMed Scopus (112) Google Scholar]. This finding confirmed the helical structure of DNA [1Watson J.D. Crick F.H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (8503) Google Scholar]. In 1979 Rich's laboratory crystallized chemically synthesized (CG)3 with a resolution of 0.9 Å [11Wang A.H. et al.Molecular structure of a left-handed double helical DNA fragment at atomic resolution.Nature. 1979; 282: 680-686Crossref PubMed Scopus (1614) Google Scholar]. As a result they discovered a new form of DNA. The zigzag folding of the polydeoxynucleotide chain (Z-DNA) was left-handed and completely different from that of the B- or A-DNA form. Although the biological role of Z-DNA is unknown, it suggests that the 3D conformation of DNA has an important role in the context of the cell. Taken together, these scientific discoveries affect our understanding of the genetic code from its transmission all the way to its translation into proteins. One should notice that many of these findings, made almost 60 years ago, were realized with very simple spectroscopic techniques (Table 1).Table 1The Great Discoveries of Alexander RichNew FindingYearRefs1Discovery of RNA double helix19562Rich A. Davies D.R. A new two-stranded helical structure: polyadenylic acid and polyuridylic acid.J. Am. Chem. Soc. 1956; 78: 3548-3549Crossref Scopus (55) Google Scholar, 3Felsenfeld G. Rich A. Studies on the formation of two- and three-stranded polyribonucleotides.Biochim. Biophys. Acta. 1957; 26: 457-468Crossref PubMed Scopus (231) Google Scholar2Nucleic acid hybridization19604Rich A. A hybrid helix containing both deoxyribose and ribose polynucleotides and its relation to the transfer of information between the nucleic acids.Proc. Natl. Acad. Sci. U.S.A. 1960; 46: 1044-1053Crossref PubMed Google Scholar3The first mention of the RNA World19626Rich A. On the problems of evolution and biochemical information transfer.in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 1962: 103-126Google Scholar4An early statement of antisense RNA19626Rich A. On the problems of evolution and biochemical information transfer.in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 1962: 103-126Google Scholar5Discovery of polyribosomes19637Warner J.R. et al.A multiple ribosomal structure in protein synthesis.Proc. Natl. Acad. Sci. U.S.A. 1963; 49: 122-129Crossref PubMed Scopus (244) Google Scholar6Single crystals of tRNA19688Kim S.H. Rich A. Single crystals of transfer RNA: an X-ray diffraction study.Science. 1968; 162: 1381-1384Crossref PubMed Scopus (47) Google Scholar7The first crystals of tRNA suitable for detailed structural analysis19719Kim S.H. et al.Three-dimensional structure of yeast phenylalanine transfer RNA: folding of the polynucleotide chain.Science. 1973; 179: 285-288Crossref PubMed Scopus (288) Google Scholar8The L-shaped folding of tRNAPhe at 3-Å resolution197410Suddath F.L. et al.Three-dimensional structure of yeast phenylalanine transfer RNA at 3.0 Ångstroms resolution.Nature. 1974; 248: 20-24Crossref PubMed Scopus (112) Google Scholar9The zigzag form of DNA (Z-DNA)197911Wang A.H. et al.Molecular structure of a left-handed double helical DNA fragment at atomic resolution.Nature. 1979; 282: 680-686Crossref PubMed Scopus (1614) Google Scholar Open table in a new tab Rich was interested in new trends across diverse disciplines. He built bridges between science and culture. Rich inspired ‘bioart’, which is an intersection between human art culture and bacterial cell culture. At his MIT laboratory, Rich had an opportunity to provide the tools of cell and molecular biology in artistic work [12Nadis S. Science for art's sake.Nature. 2000; 407: 668-670Crossref PubMed Scopus (7) Google Scholar]. As a result more artists reference facts of science and technology, a validation of the success of the project. Finally, we would like to quote a statement made by Robert Gallo about Alex Rich, with which we concur, that ‘no one else has made as many major contributions to all facets of molecular biology’.

RNA as Major Components in Chemical Evolvable Systems - Peter Strazewski How the Early Genetic Code Was Established? -Inference from the Analysis of Extant Animal Mitochondrial Decoding Systems - Kimitsuna Watanabe and Shin-ichi Yokobori Isomerization of RNA Phosphodiester Linkages - Harri Lonnberg Effects of Ionic Liquid and Liposomes on the Structure, Stability, and Function of Nucleic Acids - Naoki Sugimoto Oxidative Damage on RNA Nucleobases - Pascal A. Kupfer and Christian J. Leumann Use of FRET to Study Dynamics of DNA Replication - Philip Nevin and Penny J. Beuning Design, Characterization, and Application of Imidazopyridopyrimidine:Naphthyridine Base-Pairing Motifs Consisting of Four Hydrogen Bonds - Noriaki Minakawa and Akira Matsuda Creation of Unnatural Base Pair Systems Toward New DNA/RNA Biotechnologies - Michiko Kimoto and Ichiro Hirao Flexible Nucleobase Analogues. Novel Tools for Exploring the Nucleic Acids - Sarah C. Zimmermann and Katherine L. Seley-Radtke Sequence Selective Recognition of Double-Stranded RNA - Eriks Rozners Determining Transient Nucleic Acid Structures by NMR - Jeetender Chugh Diastereomer-Specific Repertoire of 7'R- or 7'S-Me-Carba-Locked Nucleic Acids (cLNAs) in Antisense Oligo/RNA Duplexes and Engineering of Physicochemical and Enzymological Properties - Qing Li, Oleksandr Plashkevych, Ram Shankar Upadhayaya, Sachin Gangadhar Deshpande, Andras Foldesi, and Jyoti Chattopadhyaya Challenges and Opportunities for Oligonucleotide-Based Therapeutics by Antisense and RNA Interference Mechanisms - Ramon Eritja, Montserrat Terrazas, Santiago Grijalvo, Anna Avino, Adele Alagia, Sonia Perez-Rentero, and Juan Carlos Morales Progress in Chemically Modified Nucleic Acid Aptamers - Masayasu Kuwahara Aptamers as Molecular Smugglers - Eileen Magbanua and Ulrich Hahn Biochemical Aspects of Subcellular RNA Transport and Localization - Diana Bauermeister, Maike Claussen, and Tomas Pieler Small Size, Big Impact: Bacterial Functional Nucleic Acids and Their Applications - Wendy W.K. Mok, Simon A. McManus, and Yingfu Li Towards Defined DNA and RNA Delivery Vehicles Using Nucleic Acid Nanotechnology - Anders Hauge Okholm, David Schaffert, and Jorgen Kjems Targeted Editing of Therapeutic Genes using DNA-Based Transcriptional Activators: Scope and Challenges - Ganesh N. Pandian and Hiroshi Sugiyama Interaction of DNA Intramolecular Structures with Their Complementary Strands: A Thermodynamic Approach for the Control of Gene Expression - Irine Khutsishvili, Sarah E. Johnson, Calliste Reiling, Istok Prislan, Hui-Ting Lee, and Luis A. Marky Site Directed Spin Labelling of RNA for Distance Measurements by EPR - Joachim W. Engels, Christian Grunewald, and Lena Wicke Chemo-Enzymatic Strategies to Modify RNA in vitro or in Living Cells - Daniela Schulz and Andrea Rentmeister Metal Dependence of Ligand Binding and Heavy-Atom Derivatization of Evolutionarily Distinct PreQ1 Riboswitches - Joseph E. Wedekind, Joseph A. Liberman, Jermaine L. Jenkins, and Mohammad Salim DNA G-Quadruplexes and I-Motifs in Therapeutics and Diagnostics - Yogini P. Bhavsar-Jog, Samantha M. Reilly, and Randy M. Wadkins Peptides Targeting G-Quadruplex Structures - Kenji Usui and Arisa Okada Synthesis of Site-Specifically Modified Long-Mer RNAs - Darko Balke, Jennifer Frommer, Nico Rublack, Danilo Springstubbe, Bettina Appel, and Sabine Muller Synthesis and Exon-Skipping Activity of Chemically-Modified RNAs - Yoshiaki Masaki, Takeshi Yamada, Hisao Saneyoshi, Akihiro Ohkubo, Kohji Seio, and Mitsuo Sekine mRNA and snRNA Cap Analogs: Synthesis and Applications - Janusz Stepinski and Edward Darzynkiewicz Innovative Chemistry for Synthesis of Regular RNA, 5'-Triphosphate RNA or 5'-Capped RNA - Yann Thillier, Francois Morvan, Jean-Jacques Vasseur and Francoise Debart.

In this work, the protein pattern of novel Halomonas smyrnensis AAD6T was compared to that of Halomonas salina DSMZ5928T, which is the closest species on the basis of 16S rRNA sequence, to understand how AAD6T differs from type strains. Using high resolution NEPHEGE technique, the whole cell protein composition patterns of both Halomonas salina DSMZ5928T and H. smyrnensis AAD6T were mapped. The expressed proteins of the two microorganisms were mostly located at the acidic side of the gels, at molecular weight values of 60 to 17 kDa, and at isoelectric points 3.8 to 6.0, where they share a significant number of common protein spots. Identification and characterization of protein spots via whole genome sequencing data indicated that these two microorganisms used similar pathways, especially TCA cycle, for their survival; in other words, for their energy requirements. On the other hand, the protein expression differences in AAD6T and H. salina DSMZ 5928T showed that they prefer different metabolic pathways for lipid biosynthesis and in adaptation to extreme environments. Thus, we suggested that phylogenetic dissimilarities between these microorganisms could be related to the protein expression differences; in other words, metabolic flux differences in AAD6T and H. salina DSMZ 5928T. This is the first study to explain the dissimilarities of phenotypic characters and DNA-DNA hybridization between type strain and novel strain AAD6T by using protein expression differences.

With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down.

This book will provide latest insights in the functional potentials of ribonucleic acids in medine and the use of Spiegelmer and Spiegelzyme systems. It will also deal with a new type of delivery syst

In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.

Despite a half century of structural, biophysical and biochemical investigations of ribonucleic acids, they are still mysterious. RNAs stand at fertile crossroads of disciplines, integrating concepts

Am 27. Mai 1961 führten Nirenberg und Matthaei ein In-vitro-Translationsexperiment durch, in dem bakterielle Ribosomen mit niedermolekularer RNA versetzt wurden. Dabei konnten sie zeigen, dass Polyuridylsäure für Polyphenylalanin kodiert. Diese Experimente lieferten erste Informationen über den genetischen Code. Dieser Essay erinnert an die bedeutenden Errungenschaften vor der Entdeckung des genetischen Codes, die Experimente, die zu seiner Entschlüsselung führten, und nachfolgende Entwicklungen auf dem Gebiet der Molekularbiologie.

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface. In our proof-of-principle experiment we could observe the direct folding of long, human telomeric repeats induced by the small analyte potassium using surface plasmon resonance spectroscopy. This work demonstrates the feasibility of new evanescent field-based biosensors that can specifically observe small molecule interactions.

BackgroundSELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.MethodologyWe have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel.ConclusionsHigh affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

On May 27, 1961 Nirenberg and Matthaei performed an in vitro translation experiment in which bacterial ribosomes were supplemented with a fraction of low-molecular-weight RNAs; they demonstrated that polyuridilic acid codes for polyphenylalanine—the first information on the genetic code. This Essay recalls the major achievements prior to the discovery of the genetic code, the experiments leading to its identification, and the developments in the field of molecular biology thereafter.