Supplementary Figures providing detailed experimental protocols and methodologies from Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

The supplementary figures provide details of the experimental protocols and methodologies as well as the raw data for ligation reaction

Supplementary Tables of sequence data, meme analyses and RNA structures from Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

The supplementary tables provide the raw data for oligonucleotides, sequence data, meme analyses and secondary structures of RNA molecule

Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids.

<p>A. A single blot was probed with an oligonucleotide that was complementary to putative <i>tat</i>, <i>rev</i> and <i>vif</i> guide sequences. B. The blot was probed with two 14-mer LNA oligonucleotides (LNA nucleotides underlined) which were complementary to the <i>rev</i> siRNA guide sequence and adjacent nucleotides as indicated in the illustration. Both blots (from A and B) were stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.</p

Effect of nucleotide spacing between <i>tat</i>, <i>rev</i> and <i>vif</i> siRNA-encoding sequences on silencing efficacy.

<p>A. Schematic illustration of hairpin sequences with boundaries between <i>tat</i>, <i>rev</i> and <i>vif</i> duplexes indicated. The sequences and numbers of bases inserted or deleted at the junctions of the RNAi effecter-encoding sequences are indicated for each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. B. Average normalized <i>Renilla</i>∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck <i>tat-rev-vif</i> target together with each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. C. Average normalized <i>Renilla</i>∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck <i>tat</i>, psiCheck <i>rev or</i> psiCheck <i>vif</i> target together with each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. Results are given as the average values with standard deviations from three independent transfection experiments. (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control). Mock transfected cells received the empty backbone U6+1 plasmid D. Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids. The blot was probed with an oligonucleotide that was complementary to putative <i>rev</i> guide sequence. The blot was stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.</p

HIV-1 challenge assay.

<p>U87.CD₄.CCR5 cells were transfected with plasmids expressing the indicated hairpins and then subjected to infection with an equivalent of TCID₅₀ 1000 particles of the HIV-1 FV5 viral isolate. Concentrations of HIV-1 p24 in the culture supernatant and viral particle equivalents (A) were determined 6 days after infection. Results are expressed as the means with standard deviations of three independent experiments (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control). B. Replication kinetics of a representative experiment included in (A). C. Sequence of lhRNA- and shRNA-derived <i>tat</i>, <i>rev</i> and <i>vif</i> guide sequences and complementary regions targeted within FV5 and HXB2 HIV-1 isolates. Mismatches between the putative guide and target sequences are shown. Sequence numbering is based on isolate HXB2, accession K03455. D. Knockdown of FV5 <i>vif</i> and HXB2 <i>vif</i> target-reporter fusion gene expression by shRNA <i>vif</i> and representative lhRNAs in HEK293 cells. Results are expressed as the means with standard deviations of three independent experiments (*; p<0.05, <i>t</i>-test, between annotated samples).</p

Knockdown of target-reporter fusion gene expression.

<p>A. psiCheck-derived vectors that include indicated HIV-1 target sequences inserted downstream of the <i>Renilla</i> luciferase ORF. The control Firefly luciferase cassette, present on the same plasmid, is also shown. Both cassettes are under control of constitutively active transcription regulatory elements: Herpes simplex virus thymidine kinase (HSV TK) and Simian virus 40 (SV40) promoters. B. Average normalized ratios of the <i>Renilla</i>∶Firefly luciferase activity when cells were transfected with psiCheck <i>tat</i>-<i>rev</i>-<i>vif</i> dual luciferase reporter plasmid together with lhRNA- or shRNA-encoding plasmid vectors. C. Average normalized ratios of the <i>Renilla</i>∶Firefly luciferase activity when cells were transfected with psiCheck <i>tat</i>, psiCheck <i>rev</i> or psiCheck <i>vif</i> dual luciferase reporter plasmids together with lhRNA- or shRNA-encoding plasmid vectors. The average values from three independent transfection experiments, with standard deviations, are given (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control).</p