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.