Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. ...In this work, two different diastereoisomers of LNA were studied: the beta‐d‐LNA and the alpha‐l‐LNA (abbreviated as β‐d‐LNA and α‐l‐LNA). Our findings show that the best antisense activity with 16mer gapmers containing β‐d‐LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA–DNA–LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif‐dependent, and requires the right balance between gap size and affinity. Compared to β‐d‐LNA, α‐l‐LNA shows superior stability against a 3′‐exonuclease. The design possibilities of α‐l‐LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of α‐l‐LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, α‐l‐LNA and β‐d‐LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with α‐l‐LNA still recruit RNase H and show good levels of antisense activity, while the same design with β‐d‐LNA results in a drop in antisense potency. Our findings indicate that α‐l‐LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.
Summary
Secondary amyloidosis is usually a complication of chronic inflammation. Amyloidosis cases during the course of non‐Hodgkin's lymphoma (NHL) are usually of AL‐type, only one NHL patient with ...secondary amyloidosis has been reported. Our 79‐year‐old male patient visited us with multiple lymphadenopathies, and he was diagnosed with nodal marginal zone B‐cell lymphoma. After four cycles of combined chemotherapy; his urea, creatinine levels started to increase and he developed nephrotic‐range proteinuria. His rectal biopsy demonstrated amyloid deposition in submucosal vessel walls. The patient has been under hemodialysis for 10 months and his lymphoma is still in partial remission. We presented this case because it is the second NHL patient who developed secondary amyloidosis during his disease course.
An empirical formula for thermal stability (Tm) prediction of PNA/DNA duplexes has been derived. The model is based on the Tm as calculated for the corresponding DNA/DNA duplex employing a nearest ...neighbour approach, by including terms for the pyrimidine content and length of the PNA to take into account the increased thermostability of PNA/DNA hybrids and the asymmetry of the PNA-DNA heteroduplex. The predictive power of the Tm prediction formula was challenged with an independent data set not used for model building. The Tm of >90% of the sequences was predicted within 5 K; 98% of the predicted Tms differ by not more than 10 K from the experimentally determined Tm.
We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila. The genes of the individual snRNAs exist in two to six slightly ...different copies per haploid genome. Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes. Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes. Comparison of the promoter structure of the snRNA genes of T. thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms. This finding is discussed in relation to a possible role in snRNA 3'-end formation.
Aptamers interacting with RNA hairpins through loop−loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA ...aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.
A nucleolar snRNA previously proposed to be the T. thermophila homologue of mammalian U3-snRNA was isolated by preparative polyacrylamide get electrophoresis and sequenced by a combination of ...established methods. The sequence identified two very similar RNA species of identical size (256 nt.) and with unique 5' and 3' ends.
One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome ...this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV‐1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV‐spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.
We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate,
Tetrahymena. The snoRNAs are 120–140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their ...5′ ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.
We have previously characterized the T. thermophila homologues of the major mammalian U-snRNAs, U1 to U6. During this work a new snRNA species was reproductively recovered in the nuclear fraction. ...The sequence of this snRNA was established by direct chemical sequencing followed by dideoxy-sequencing using primers complementary to the chemically determined sequence at the 3' end. Furthermore, the 3' end of the snRNA was mapped by the previously described reverse dideoxy sequencing method. The combined sequence data defines and snRNA with a total size of 99 nt with unique 5' and 3' ends. We have termed this snRNA Tx-1.
Radioactive labelling of PNA has been performed by linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields.
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