Association of small vessel disease with tau pathology.

Emerging evidence suggests that small vessel disease (SVD) is a risk factor for clinical dementia and may contribute to AD neuropathological changes. Watershed brain regions are located at the most distal areas between arterial territories, making them vulnerable to SVD-related changes. We examined the association of pathologic markers of SVD, specifically arteriolosclerosis in watershed brain regions, with AD pathologic changes. Participants (N = 982; mean age-at-death = 90; 69% women) were enrolled as part of one of two cohort studies of aging and dementia. At autopsy, neuropathological evaluation included semi-quantitative grading of arteriolosclerosis pathology from 2 cortical watershed regions: the anterior watershed (AWS) and posterior watershed (PWS), densities for cortical ß-amyloid and tau-tangle pathology, and other common age-related pathologies. Linear regression models examined the association of watershed arteriolosclerosis pathology with ß-amyloid and tau-tangle burden. In follow-up analyses, available ex-vivo MRI and proteomics data in a subset of decedents were leveraged to examine the association of whole brain measure of WMH, as a presumed MRI marker of SVD, with ß-amyloid and tau-tangle burden, as well as to examine the association of watershed arteriolosclerosis with proteomic tau. Watershed arteriolosclerosis was common, with 45% of older persons having moderate-to-severe arteriolosclerosis pathology in the AWS region, and 35% in the PWS. In fully adjusted models that controlled for demographics and common age-related pathologies, an increase in severity of PWS arteriolosclerosis was associated with a higher burden of tau-tangle burden, specifically neocortical tau burden, but not with ß-amyloid. AWS arteriolosclerosis was not associated with ß-amyloid or tau pathology. Ex-vivo WMH was associated with greater tau-tangle pathology burden but not ß-amyloid. Furthermore, PWS arteriolosclerosis was associated with higher abundance of tau phosphopeptides, that promote formation of tau aggregates. These data provide compelling evidence that SVD, specifically posterior watershed arteriolosclerosis pathology, is linked with tau pathological changes in the aging brain.

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure.

Oligonucleotide conjugates bearing two pyrene residues attached to 5'-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (lambda(max) 382 nm) and a broad emission peak with lambda(max )476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5'-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5'-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.

Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe).

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.

Invasion of strongly binding oligonucleotides into tRNA structure.

Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2'-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65-76 of the tRNA(Phe).

Mechanism of antisense oligonucleotide interaction with natural RNAs.

Oligonucleotides find several numbers of applications: as diagnostic probes, RT and PCR primers and antisense agents due to their ability of forming specific interactions with complementary nucleotide sequences within nucleic acids. These interactions are strongly affected by accessibility of the target sequence in the RNA structure. In the present work the mechanism of invasion of RNA structure by oligonucleotide was investigated using a model system: yeast tRNA(Phe) and oligonucleotides complementary to the 3'-part of this molecule. Kinetics of interaction of oligonucleotides with in vitro transcript of yeast tRNAPhe was studied using stopped-flow technique with fluorescence quenching detection, 5'-DABCYL labeled oligonucleotide was hybridized with 3'-fluorescein labeled tRNA(Phe). The results evidence for a four-step invasion process of the oligonucleotide-RNA complex formation. The process is initiated by formation of transition complexes with nucleotides in the T-loop and ACCA sequence. This complex formation is followed by RNA unfolding and formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Computer modeling of oligonucleotide-tRNA(Phe) interaction revealed potential factors that could favor transition complexes formation and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular "wedge". Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA-RNA interactions. The obtained results can be used for choosing efficient oligonucleotide probes.

Interaction of complementary oligonucleotides with the 3'-end of yeast tRNA(Phe).

Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides (ONs), complementary to the nucleotides 62-76 was investigated. Results of gel-mobility shift assay and RNase A probing evidence that the ONs containing the sequence complementary to the tRNA ACCA end can easily invade the hairpin structure under physiological conditions. The limiting step of association process is the tRNA unfolding.