New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3′ end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells

New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3′ end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells

Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells

Synthetic RNA formulations and viral vectors are the two main approaches for delivering small therapeutic RNA to human cells. Here we report findings supporting an alternative strategy in which an endogenous human RNA polymerase (RNAP) is harnessed to make RNA hairpin-containing small RNA from synthetic single-stranded DNA oligonucleotides. We report that circularizing a DNA template strand encoding a pre-microRNA hairpin mimic can trigger its circumtranscription by human RNAP III in vitro and in human cells. Sequence and secondary structure preferences that appear to promote productive transcription are described. The circular topology of the template is required for productive transcription, at least in part, to stabilize the template against exonucleases. In contrast to bacteriophage and Escherichia coli RNAPs, human RNAPs do not carry out rolling circle transcription on circularized templates. While transfected DNA circles distribute between the nucleus and cytosol, their transcripts are found mainly in the cytosol. Circularized oligonucleotides are synthetic, free of the hazards of viral vectors and maintain small RNA information in a stable form that RNAP III can access in a cellular context with, in some cases, near promoter-like precision and biologically relevant efficiency

Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

Single-stranded (ss) circular oligodeoxynucleotides were previously found to undergo rolling circle transcription (RCT) by phage and bacterial RNA polymerases (RNAPs) into tandemly repetitive RNA multimers. Here, we redesign them to encode minimal primary miRNA mimics, with the long term aim of intracellular transcription followed by RNA processing and maturation via endogenous pathways. We describe an improved method for circularizing ss synthetic DNA for RCT by using a recently described thermostable RNA ligase, which does not require a splint oligonucleotide to juxtapose the ligating ends. In vitro transcription of four templates demonstrates that the secondary structure inherent in miRNA-encoding vectors does not impair their RCT by RNAPs previously shown to carry out RCT. A typical primary-miRNA rolling circle transcript was accurately processed by a human Drosha immunoprecipitate, indicating that if human RNAPs prove to be capable of RCT, the resulting transcripts should enter the endogenous miRNA processing pathway in human cells. Circular oligonucleotides are therefore candidate vectors for small RNA delivery in human cells, which express RNAPs related to those tested here

Rolling Circle Transcription (RCT) approach to expressing miRNA, siRNA and other small RNA.

<p><b>A</b>. Circular oligodeoxynucleotides (COLIGOs) are made to encode minimal pri-miRNAs. RCT would produce tandemly arrayed primary miRNA stem-loops resembling naturally occurring miRNA clusters to promote entry into the endogenous miRNA maturation pathway, and Argonaute effector complexes (RISC) programming. <b>B</b>. RNA transcript sequences and the predicted secondary structures of the four COLIGOs used in this study. Mature miRNA are shaded, number in parentheses refers to number of nucleotides in COLIGO and its monomeric transcript, which is shown arbitrarily beginning outside of the stem-loop.</p

<i>In vitro</i> Drosha processing of miRNA Rolling Circle Transcripts (RCT).

<p><b>A</b>. Deproteinized miR-19am RCTs made <i>in vitro</i> using <i>E. coli</i> RNA polymerase were incubated with HEK293T whole cell extract (WCE, Lane 2); with the Flag-Drosha complex immunoprecipitated from Flag-Drosha-expressing HEK293T WCE (Lane 4); or with Flag immunoprecipitate from WCE made from untransfected HEK293T cells (Mock, Lane 3). 90 min. processing reactions. <b>B</b>. Drosha processing reactions at various processing times using DPAGE gel purified miR-19am RCT transcripts (lanes 5–9) or the miR-23a∼27a∼24-2 cluster transcript as a positive control (lanes 10–12) at the standard 90 min. processing reaction time.</p

<i>In vitro</i> transcription of miRNA-encoding COLIGOs by <i>E. coli</i> and bacteriophage T7 RNA polymerases.

<p><b>A</b>. Linear (L) and circular (C) templates 122, 122m, 19a, 19am, encoding either pre-miR-122 or pre-miR-19a, were transcribed in the presence of all NTPs or all except ATP (C-). 9% DPAGE. The Phosphorimager sensitivity setting was increased 5-fold for the weaker T7 RCT reactions. <b>B</b>. Denaturing 1% agarose gel showing the high molecular weight size range of the transcripts. No adjustment in the Phosphorimager sensitivity setting was made.</p

Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription.

<p><b>A</b>. Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. <b>B</b>. Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. <b>C</b>. Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.</p

The imprinted Air ncRNA is an atypical RNAPII transcript that evades splicing and escapes nuclear export

Expression of the Air ncRNA is necessary to silence multiple genes in cis in the imprinted Igf2r cluster. However, its mode of action is unknown. Here, we characterize co- and post-transcriptional features of Air that identify it as a new member of the class of nuclear regulatory RNAs. We show that Air is transcribed from a DNA methylation-sensitive promoter by RNA polymerase II (RNAPII). However, although it is capped and polyadenylated similar to other RNAPII transcripts, the majority of Air transcripts evade cotranscriptional splicing resulting in a mature 108 kb ncRNA. As a consequence, the mature unspliced Air is nuclear localized and highly unstable. These features show that Air is an atypical RNAPII transcript whose properties indicate that its mode of action in gene silencing may not depend on the RNA per se but instead is related to its actual transcription