Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation.

Stress insults intensify fear memory; however, the mechanism(s) facilitating this physiological response is still unclear. Here, we report the molecular, neurophysiological and behavioral findings attributing much of this effect to alternative splicing of the acetylcholinesterase (AChE) gene in hippocampal neurons. As a case study, we explored immobilization-stressed mice with intensified fear memory and enhanced long-term potentiation (LTP), in which alternative splicing was found to induce overproduction of neuronal 'readthrough' AChE-R (AChE-R). Selective downregulation of AChE-R mRNA and protein by antisense oligonucleotides abolished the stress-associated increase in AChE-R, the elevation of contextual fear and LTP in the hippocampal CA1 region. Reciprocally, we intrahippocampally injected a synthetic peptide representing the C-terminal sequence unique to AChE-R. The injected peptide, which has been earlier found to exhibit no enzymatic activity, was incorporated into cortical, hippocampal and basal nuclei neurons by endocytosis and retrograde transport and enhanced contextual fear. Compatible with this hypothesis, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKCbetaII, accompanied by PKC-augmented LTP enhancement. Our findings demonstrate a primary role for stress-induced alternative splicing of the AChE gene to elevated contextual fear and synaptic plasticity, and attribute to the AChE-R splice variant a major role in this process.

Cyclohexene nucleic acids (CeNA) form stable duplexes with RNA and induce RNase H activity.

Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against degradation in serum. A CeNA/RNA hybrid is able to activate E. Coli RNase H. resulting in cleavage of the RNA strand.

RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA).

Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA-RNA duplex (DeltaTm per modification: +2 degrees C). A cyclohexenyl A nucleotide adopts a 3'-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4'-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The Vmax and Km of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the kcat value is about 600 times lower in the case of CeNA:RNA.

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.

The alpha-L-ribo-isomers of RNA and LNA (locked nucleic acid).

The phosphoramidite approach has been used for the automated synthesis of alpha-L-LNA, alpha-L-RNA, and oligomers composed of mixtures of alpha-L-LNA, alpha-L-RNA and DNA monomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. alpha-L-LNAs were shown to be significantly stabilized towards 3'-exonucleolytic degradation. Duplexes formed between RNA and alpha-L-LNA induced E. coli RNase H-mediated RNA cleavage, albeit very slow, at high enzyme concentration.