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Circularized single-stranded DNA oligonucleotides, or coligos, show promise as promoter-independent RNA polymerase III (Pol III) transcription templates for generating small RNA in human cells. Using a modified small RNA-seq method, we studied the sequence and secondary structure characteristics that determine Pol III initiation and termination on six coligo templates. The coligos each consisted of an imperfectly base-paired stem flanked by one larger and one smaller loop and were unrelated in sequence. Small RNA-seq data from Pol III coligo transcripts revealed a strong preference for initiating transcription within a 5-nucleotide (nt) window spanning the stem-larger loop junction (loop size 11–24 nt). Transcription in all cases proceeded into the stem rather than into the larger loop, indicating the junction is a site-specific, secondary structure-based Pol III transcription initiator. On average, 81% of sequencing reads showed initiation within this 5 nt junction region, with a template start site nucleotide preference of C > T >> A > G, and a requirement for a template purine at Tss-1. Termination was less precise than initiation and occurred in the larger loop at the same end of the stem where transcription initiated. Termination efficiency was on average 82% and was distributed among the first 11 single-stranded larger loop nt following the stem. The size heterogeneity of Pol III coligo transcripts is thus mainly due to 3′ end heterogeneity, whereas the RNA 5′ ends were more predictable and homogeneous. Transcription termination did not require an oligo dA template sequence, indicating that termination in this context may be mechanistically different than Pol III's normal gene-context termination. A stepwise model for coligo transcription by Pol III is proposed.

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.

Many high-throughput small RNA next-generation sequencing protocols use 5′ preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5′ end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5′ adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3′ blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3′ ends to be 5′ adenylylated at >90% efficiency.

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.

The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5’ recessed ends are poor substrates for enzymatic adapter ligation, but this 5’ adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5’ RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5’ ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5’ adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5’ end. This method accepts a variety of 5’ modified RNA, including 5’ monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library.

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.