Studies of Impending Oligonucleotide Therapeutics in Simulated Biofluids.

Synthetic oligonucleotides, their complexes and conjugates with other biomolecules represent valuable research tools and therapeutic agents. In spite of growing applications in basic research and clinical science, only few studies have addressed the issue of such compounds' stability in biological media. Herein, we studied the stability of two therapeutically relevant oligonucleotide probes in simulated biofluids; the 21 nucleotide-long DNA/locked nucleic acid oligonucleotide ON targeted toward cancer-associated BRAF V600E mutation, and a longer DNA analog (TTC) originating from BRAF gene.

Mapping of ribosomal 23S ribosomal RNA modifications in Clostridium sporogenes.

All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA.

Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr.

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation.

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides.

Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases.

Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor.

A cfr-like gene from Clostridium difficile confers multiple antibiotic resistance by the same mechanism as the cfr gene.

The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostridium difficile) strain does function as a Cfr protein.

Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance.

Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance.

Improvement of a streptavidin-binding aptamer by LNA- and α-l-LNA-substitutions.

Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and compared with the unmodified DNA aptamers. A ∼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3'-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability.

A click chemistry approach to pleuromutilin derivatives. Part 3: extended footprinting analysis and excellent MRSA inhibition for a derivative with an adenine phenyl side chain.

Five promising pleuromutilin derivatives from our former studies, all containing adenine on various linkers, were supplemented with two new compounds. The binding to Escherichia coli ribosomes was verified by extensive chemical footprinting analysis. The ability to inhibit bacterial growth was investigated on two Staphylococcus aureus strains and compared to the pleuromutilin drugs tiamulin and valnemulin.

Distinction between the Cfr methyltransferase conferring antibiotic resistance and the housekeeping RlmN methyltransferase.

The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the RlmN methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance.

Peptide-LNA oligonucleotide conjugates.

Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry.

Amplification and re-generation of LNA-modified libraries.

Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.

The order Bacillales hosts functional homologs of the worrisome cfr antibiotic resistance gene.

The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisome cfr gene and functionally similar genes.

A click chemistry approach to pleuromutilin derivatives, part 2: conjugates with acyclic nucleosides and their ribosomal binding and antibacterial activity.

Pleuromutilin is an antibiotic that binds to bacterial ribosomes and thereby inhibit protein synthesis. A new series of semisynthetic pleuromutilin derivatives were synthesized by a click chemistry strategy. Pleuromutilin was conjugated by different linkers to a nucleobase, nucleoside, or phenyl group, as a side-chain extension at the C22 position of pleuromutilin.

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library.

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences.

Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution.

G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations.

Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX(3)CX(2)C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes.

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp.

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification.

A click chemistry approach to pleuromutilin conjugates with nucleosides or acyclic nucleoside derivatives and their binding to the bacterial ribosome.

Pleuromutilin and its derivatives are antibacterial drugs that inhibit protein synthesis in bacteria by binding to ribosomes. To promote rational design of pleuromutilin based drugs, 19 pleuromutilin conjugates with different nucleoside fragments as side chain extensions were synthesized by a click chemistry protocol. Binding was assessed by chemical footprinting of nucleotide U2506 in 23S rRNA, and all conjugates bind to varying degree reflecting their binding affinity to the peptidyl transferase center.

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets.

Background: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.

Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting.

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start.

The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double-stranded DNA (dsDNA, 26.0 degrees C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.

A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503.

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant.