RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry

Abstract

Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III.

Starting and stopping RNA polymerase III transcription on single-stranded DNA oligonucleotides

“…As with 15a-3, transcription initiation on mut2B occurred primarily at a single dC nucleotide near the stem-larger loop junction, though in this case the dC was located on the stem side of the junction. Note that transcription initiation at the junction is site-specific, always proceeding into the stem, and never from the stem into the loop, consistent with our structural prediction of a unidirectional alignment of the coligo within the Pol III active site at initiation ( Patel et al 2016 ). Also similar to 15a-3, transcript size heterogeneity was due to 3′ end heterogeneity, and termination was distributed among the first ten or eleven larger loop nucleotides.…”

“…3 ): 15a-3 5′G C A > T T G; mut2B G C G > A T A; 122 A T A > T T G > T T T; 221 C T G > T T G > G T T; 143-2 A C A > T A A; Dcr3 G C A > C A A. The stem should also have a small symmetrical internal loop within 8 bp of the junction ( Lama et al 2014 ), as in 15a-3, mut2B, 122 and 143-2; the larger loop should be ≥10–11 nt; and the smaller loop should not exceed ∼5–6 nt ( Patel et al 2016 ).…”

“…We are investigating c ircularized synthetic DNA oligo nucleotide s , or coligos, as potential vectors for engineering the expression of small RNA in human cells. As expression vectors, coligos are unique in that they contain only the template strand and lack a transcription promoter sequence, instead appearing to rely on sequence- and secondary structure-triggered transcription initiation and termination ( Seidl and Ryan 2011 ; Seidl et al 2013 ; Lama et al 2014 ; Patel et al 2016 ). Coligos lack 5′ and 3′ ends, a feature that reduces susceptibility to nucleases and further distinguishes them structurally from other promoter-independent eukaryotic transcription templates like the 3′ overhang and oligo dC-tailed templates ( Dedrick and Chamberlin 1985 ; Campbell and Setzer 1992 ; Bardeleben et al 1994 ; Schroder et al 2003 ).…”

“…To encode this hairpin RNA motif, the coligos contain an imperfectly base-paired stem flanked by one larger and one smaller loop, and thus resemble asymmetric DNA dumbbells ( Erie et al 1987 ). Pre-miRNA-encoding coligos generally produce hairpin transcripts with modest to very good end heterogeneity, as judged by electrophoresis and preliminary cDNA sequencing analyses ( Seidl et al 2013 ; Lama et al 2014 ; Patel et al 2016 ). Most coligo-dependent transcripts result from a single circumtranscription around the template, with in most cases negligible rolling circle transcription.…”

Impact of foliar application of carbon nanotube and benzyladenine on broccoli growth and head yield

“…After their entry, nanomaterials (NMs) reach the tissues of the epidermis and the mesophyll where they can interact with these tissues and their structures, and later they can be translocated to the whole plant through the vascular bundles (Hubbard et al, 2020). In addition, CNTs act as elicitors in the regulation of plant growth (Patel et al, 2017), since they activate the biosynthesis of indole acetic acid and abscisic acid (Cheng et al, 2016). They also promote the expression of marker genes of cell division (CycB) and elongation of the cell wall (NtLRX1) that directly influence growth, hence productivity (Khodakovskaya et al, 2012).…”