Achiral, acyclic nucleic acids: synthesis and biophysical studies of a possible prebiotic polymer.

The search for prebiotic, nucleic acid precursors is, at its best, a speculative undertaking. Given the complex structure of RNA, it is not very likely that RNA was the first information system in the universe and thus finding possible precursor/s i.e. pre-RNA remains an open challenge. We, in this paper, have tried to construct nucleic acid polymers with a simple acyclic, achiral backbone. Such a linear, achiral backbone may have been formed from simple monomers that may have existed in the "prebiotic soup". We have shown that such polymers are capable of identifying the complementary "other self" and thus forming a potential system for information storage and transmission. This study thus involves investigation of nucleic acid analogues with a modified backbone that are likely to have formed in the prebiotic setting.

Hybridization potential of oligonucleotides comprising 3'-O-methylated altritol nucleosides.

A series of 3'-O-methylated-d-altrohexitol nucleoside analogs (MANA) was synthesized comprising all four base moieties, adenine, cytosine, uracil, and guanine. These monomers were incorporated into oligonucleotides (ONs) by automated solid phase synthesis and the thermal and thermodynamic stability of all new modified constructs were evaluated. Data were compared with results obtained for both anhydrohexitol (HNAs) and 3'-O-altrohexitol-modified ONs (ANAs). We hereby demonstrate that ONs modified with MANA monomers have an improved thermal and thermodynamic stability compared to RNA, ANA, or HNA containing ONs of which the extent depends on the number of incorporated moieties and their position in the sequence. Thermodynamic analysis afforded comparable or even improved results in comparison with the incorporation of locked nucleic acids. While the specificity of these new synthons is slightly lower compared to mismatches within RNA double strands, it is similar to the discrimination potential of other hexitol modifications (HNA and ANA) which already proved their biologic interest, highlighting the potential of MANA constructs in antisense and in siRNA applications.

Antibacterial 5'-O-(N-dipeptidyl)-sulfamoyladenosines.

The aminoacyl-tRNA synthetase (aaRS) class of enzymes is a validated target for antimicrobial development. Aminoacyl analogues of 5'-O-(N-L-aminoacyl)-sulfamoyladenosines are known to be potent inhibitors of aaRS, but whole cell antibacterial activity of these compounds is very limited, and poor penetration into bacteria has been proposed as the main reason for this. Aiming to find derivatives that better penetrate bacteria, we developed a simple and short method to prepare dipeptidyl-derivatives of 5'-O-(N-L-aminoacyl)-sulfamoyladenosines, and used this method to prepare 18 5'-O-(N-dipeptidyl)-sulfamoyladenosines. The antibacterial activity of these derivatives and a number of reference compounds against S. aureus, E. faecalis and E. coli was determined. Several of the new derivatives showed improved antibacterial activity and an altered spectrum of antibacterial activity.

Chemical incorporation of 1-methyladenosine, minor tRNA component, into oligonucleotides.

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.

Methylated hexitol nucleic acids, towards congeners with improved antisense potential.

In an effort to further improve the hybridisation potential of anhydro-hexitol nucleoside analogues, the 1'-methoxyl and 3'-methoxyl substituents were introduced and evaluated for their antisense potential. In view of the selectivity of pairing with RNA, especially the introduction of a 3'-O-alkyl moiety deserves further study.

Improved hybridisation potential of oligonucleotides comprising O-methylated anhydrohexitol nucleoside congeners.

The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.

Increased RNA affinity of HNA analogues by introducing alkoxy substituents at the C-1 or C-3 position.

1,5-Anhydrohexitol nucleoside congeners with alkoxy substituents, were prepared, resulting in a further improvement of their RNA affinity and antisense potential.

Hybridization between "six-membered" nucleic acids: RNA as a universal information system.

Within the polyA:polyT recognition system, cross-pairing between several nucleic acids with a phosphorylated six-membered carbohydrate (mimic) as repeating unit in the backbone structure has been observed. All investigated nucleic acids (except for beta-homo-DNA) hybridize with RNA, leaving RNA as a versatile biopolymer for informational transfer. [reaction: see text]

Alpha-homo-DNA and RNA form a parallel oriented non-A, non-B-type double helical structure.

Cross-talking between nucleic acids is a prerequisite for information transfer. The absence of observed base pairing interactions between pyranose and furanose nucleic acids has excluded considering the former type as a (potential) direct precursor of contemporary RNA and DNA. We observed that alpha-pyranose oligonucleotides (alpha-homo-DNA) are able to hybridize with RNA and that both nucleic acid strands are parallel oriented. Hybrids between alpha-homo-DNA and DNA are less stable. During the synthesis of alpha-homo-DNA we observed extensive conversion of N6-benzoyl-5-methylcytosine into thymine under the usual deprotection conditions of oligonucleotide synthesis. Alpha-homo-DNA:RNA represents the first hybridization system between pyranose and furanose nucleic acids. The duplex formed between alpha-homo-DNA and RNA was investigated using CD, NMR spectroscopy, and molecular modeling. The general rule that orthogonal orientation of base pairs prevents hybridization is infringed. NMR experiments demonstrate that the base moieties of alpha-homo-DNA in its complex with RNA, are equatorially oriented and that the base moieties of the parallel RNA strand are pseudoaxially oriented. Modeling experiments demonstrate that the duplex formed is different from the classical A- or B-type double stranded DNA. We observed 15 base pairs in a full helical turn. The average interphosphate distance in the RNA strand is 6.2 A and in the alpha-homo-DNA strand is 6.9 A. The interstrand P-P distance is much larger than found in the typical A- and B-DNA. Most helical parameters are different from those of natural duplexes.

Synthesis and properties of O-beta-D-ribofuranosyl-(1"-2')-adenosine-5"-O-phosphate and its derivatives.

The synthesis of O-beta-D-ribofuranosyl-(1"-2')-adenosine-5"-O-phosphate and its suitably protected derivative for oligonucleotide synthesis have been developed.

New techniques for the rapid characterization of oligonucleotides by mass spectrometry.

Recent advances in combined HPLC/electrospray ionization-mass spectrometry provide effective new capabilities for the rapid characterization of oligonucleotides. Accurate mass measurements with errors < 0.3 Da, and determination of base and sugar modification and of nearest neighbor identities, can be routinely carried out on 10-100 component mixtures of RNA or DNA. These procedures are widely applicable in structural and analytical studies involving mixtures of oligonucleotides.

Determination of nearest neighbors in nucleic acids by mass spectrometry.

The identification of nearest-neighbor residues in nucleic acids provides useful constraints on establishment of base composition and sequence and is potentially applicable to a range of structural problems involving synthetic and natural polynucleotides. A new approach to this problem using electrospray ionization tandem mass spectrometry is based on measurement of precursor-product relationships derived from small fragment ions produced in the high-pressure ionization ("nozzle-skimmer") region of the instrument. Measured mass values of dinucleotide or other fragments, which give rise to mononucleotide ions N formed in the collision cell and transmitted by the second mass analyzer, establish the identities of residues adjacent to N. The technique is applicable to RNA and DNA, whether modified, or not, and is demonstrated using modified residues in nucleic acids up to the size of intact tRNA (76-mer). By monitoring of selected ion reaction channels, the method has been extended to LC/MS and to nearest-neighbor determinations directly in oligonucleotide mixtures.

The RNA Modification Database: 1999 update.

The RNA Modification Database (http://medlib.med.utah.edu/RNAmods/) provides a comprehensive listing of naturally modified nucleosides in RNA. Each file includes: chemical structure; common name and symbol; type(s) of RNA in which found and corresponding phylogenetic distribution; Chemical s registry number and index name; and initial literature citations for structure characterization and chemical synthesis. New features include capability to search database files by name or substructural features, modifications in tmRNA, and links to related data and sites.

5-Substituted pyrimidines with a 1,5-anhydro-2, 3-dideoxy-D-arabino-hexitol moiety at N-1: synthesis, antiviral activity, conformational analysis, and interaction with viral thymidine kinase.

A new series of anhydrohexitol nucleosides are described. These compounds have a pyrimidine base moiety substituted in the 5-position with a chloro (1b), trifluoromethyl (1c), vinyl (1d), 2-thienyl (1e), ethynyl (1f) or propynyl (1g) substituent. The vinyl, propynyl, and, in particular, the 5-trifluoromethyl analogue showed potent activity against herpes simplex virus (HSV), 1c with a selectivity index of >16000 against HSV-1 and >1000 against HSV-2. Conformational analysis of anhydrohexitol nucleosides using computational methods indicates that these nucleosides occur in an equilibrium between the C1 and 1C form with a DeltaE of 5.9 kJ/mol. When the anhydrohexitol nucleoside is cocrystallized with the HSV-1 thymidine kinase it adopts a 1C conformation, which is opposite to the conformation found for the small molecule alone. The enzyme, apparently, induces a conformational change, and conformational flexibility of an anhydrohexitol nucleoside may be advantageous for recognition by viral enzymes.

N6-cyclopentyl-3'-substituted-xylofuranosyladenosines: a new class of non-xanthine adenosine A1 receptor antagonists.

The present study explores the C-3' site of the 3-deoxy-3-xylofuranosyl ring of nucleoside analogues with an adenine or N6-cyclopentyladenine (CPA) base moiety and evaluates the effect on adenosine receptor affinity. Two series of sugar-modified adenosines, i.e., 3'-amido-3'-deoxyadenosines and 3'-amidated 3'-deoxyxylofuranosyladenines, were synthesized and tested for their affinity at A1 and A2a receptors in rat brain cortex and rat striatum, respectively. The modest affinity found in the "xylo series" prompted us to synthesize the corresponding N6-cyclopentyl derivatives, which proved to be well accommodated by the A1 receptors with potencies in the lower nanomolar range. This represents a new perspective in the purinergic field. The absence of a GTP-induced shift, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP indicates an antagonistic behavior of this new class of CPA analogues.

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.

Screening a random pentapeptide library, composed of 14 D-amino acids, against the COOH-terminal sequence of fructose-1,6-bisphosphate aldolase from Trypanosoma brucei.

A random pentapeptide library composed of 14 D-amino acids, including two unusual amino acids, thus representing 537,824 different peptide sequences anchored on polystyrene beads was created with each bead bearing a single pentapeptide sequence. This library was used for affinity screening against the fructose-1, 6-bisphosphate aldolase of Trypanosoma brucei labeled with biotin as well as versus the COOH-terminal labeled with fluorescein isothiocyanate. The thus selected peptide beads were identified and the appropriate sequences synthesized as peptide amides and evaluated for enzyme activity inhibition. Screening against the whole enzyme did not result in selection of an enzyme inhibitor. However, we demonstrate here that screening against a part of the enzyme involved in the catalytic activity may lead to the discovery of an enzyme inhibitor as well as an enzyme activator. Two low affinity inhibitors, RRVKF-NH2 and KThiKAR-NH2, with an IC50 of approximately 1 mM and approximately 0.2 mM, respectively, were identified. Two other pentapeptides with the sequence SWChaKK-NH2 and SKChaKM-NH2 are able to activate the enzyme fructose-1, 6-bisphosphate aldolase. Thus, successful screening of solid phase libraries can be accomplished using selected sequences of the target enzyme.

alpha-Amino acids derived from ornithine as building blocks for peptide synthesis.

Recently great interest has arisen in the synthesis of combinatorial libraries, and this technology provides a significant partner to contemporary strategies in rational design and lead discovery. By simple combination of a given set of building blocks, high numbers of different molecules are produced simultaneously, increasing the possibility of discovery of a lead compound in a limited time. One direction of research in this field focuses on the synthesis of libraries composed of modified amino acids. Here, the synthesis and characteristics of some building blocks derived from ornithine are described. The synthesis is based on the acylation/sulfonation of the copper complex of ornithine by aroyl and arylsulfonyl chlorides exemplified by 2-thiophenecarbonyl chloride, p-toluenesulfonyl chloride and 8-quinolinesulfonyl chloride. To evaluate the potential use of these modified alpha-amino acids as component in an oligopeptide library, all three derivatives were incorporated in a hexapeptide with a random sequence using a standard coupling procedure (DIC/HOBt/DIEA). Depending upon the acidity of the amido hydrogen on the delta-nitrogen, competition between intramolecular cyclization and peptide bond formation was observed. The higher the acidity, the more pronounced is this side reaction. Coupling conditions for peptide formation were optimized so that the newly described amino acid based building blocks are suitable for incorporation into libraries consisting of unnatural amino acids. The outlined procedures open up a broad avenue of possibilities for creation of diversity into peptidic libraries.

Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry.

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3'- and 5'- terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates. Algorithms for sequence derivation are embodied in a computer program that requires < 2s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direct sequencing of components of mixtures is shown using an unresolved fraction of unknown oligonucleotides from ribosomal RNA.

An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.