Background: The performance of alignment programs is traditionally tested on sets of protein sequences, of which a reference alignment is known. Conclusions drawn from such protein benchmarks do not necessarily hold for the RNA alignment problem, as was demonstrated in the first RNA alignment benchmark published so far. For example, the twilight zone – the similarity range where alignment quality drops drastically – starts at 60 % for RNAs in comparison to 20 % for proteins. In this study we enhance the previous benchmark. Results: The RNA sequence sets in the benchmark database are taken from an increased number of RNA families to avoid unintended impact by using only a few families. The size of sets varies from 2 to 15 sequences to assess the influence of the number of sequences on program performance. Alignment quality is scored by two measures: one takes into account only nucleotide matches, the other measures structural conservation. The performance order of parameters – like nucleotide substitution matrices and gap-costs – as well as of programs is rated by rank tests. Conclusion: Most sequence alignment programs perform equally well on RNA sequence sets with high sequence identity, that is with an average pairwise sequence identity (APSI) above 75 %. Parameters for gap-open and gap-extension have a large influence on alignment quality lower than APSI ≤ 75 %; optimal parameter combinations are shown for several programs. The use of different 4 × 4 substitution matrices improved program performance only in some cases. The performance of iterative programs drastically increases with increasing sequence numbers and/or decreasing sequence identity, which makes them clearly superior to programs using a purely non-iterative, progressive approach. The best sequence alignment programs produce alignments of high quality down to APSI > 55 %; at lower APSI the use of sequence+structure alignment programs is recommended.

To defend against invading pathogens, plants possess RNA silencing mechanisms involving small RNAs (miRNAs, siRNAs). Also viroids – plant infectious, non-coding, unencapsidated RNA – cause the production of viroid-specific small RNAs (vsRNA), but viroids do escape the cytoplasmic silencing mechanism. Viroids with minor sequence variations can produce different symptoms in infected plants, suggesting an involvement of vsRNAs in symptom production. We analyzed by deep sequencing the spectrum of vsRNAs induced by the PSTVd strain AS1, which causes strong symptoms such as dwarfing and necrosis upon infection of tomato plants cv Rutgers. Indeed, vsRNAs found with highest frequency mapped to the pathogenicity-modulating domain of PSTVd, supporting an involvement of vsRNAs in symptom production. Furthermore, in PSTVd AS1-infected plants the accumulation of some endogenous miRNAs, which are involved in leaf development via regulation of transcription factors, is suppressed. The latter finding supports the hypothesis that a miRNA-dependent (mis)regula-tion of transcription factors causes the viroid symptoms.

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (−)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (−)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (−)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates—similar to in vivo rates—or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.

Viroids are the smallest autonomous infectious nucleic acids known today. They are non-coding, unencapsidated, circular RNAs with sizes ranging from 250 to 400 nucleotides and infect certain plants. These RNAs are transcribed by rolling-circle mechanisms in the plant host’s nuclei (Pospiviroidae) or chloroplasts (Avsunviroidae). Since viroids lack any open reading frame, their pathogenicity has for a long time been a conundrum. Recent findings, however, show that viroid infection is associated with the appearance of viroid-specific small RNA (vsRNA). These have sizes similar to endogenous small interfering RNA and microRNA and thus might alter the normal gene expression in the host plant. In this review we will summarize the current knowledge on vsRNA and discuss the current hypotheses how they connect to the induced symptoms, which vary dramatically, depending on both the plant cultivar and the viroid strain.

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.