This study describes a method for synthesizing small, defined-length RNAs using T7 RNA polymerase and synthetic DNA templates containing the T7 promoter. The method allows for the production of RNA fragments ranging from 12 to 35 nucleotides in length, with milligram quantities achievable through optimized reaction conditions. The synthetic DNA templates, which are partially single-stranded and base-paired only in the promoter region, are as effective as linear plasmid DNA in transcription. Runoff transcripts initiate at a predictable position but may have one nucleotide more or less on the 3' end. The reactions also produce smaller oligoribonucleotides (2-6 nucleotides) as a result of abortive initiation events. Variants in the +1 to +6 region of the promoter can increase the variety of RNAs produced but reduce transcription efficiency. The study also shows that the non-template strand is not required for efficient transcription if the promoter region is double-stranded. The results indicate that the T7 RNA polymerase can add a non-template encoded nucleotide at the 3' end of the transcripts. The study further demonstrates that the length of the promoter does not significantly affect transcription efficiency, and that the T7 promoter is highly conserved from -17 to +6. The study also shows that the sequence beyond +2 has little effect on the yield of the reaction, while substitutions at +2 can reduce the yield. The study concludes that T7 RNA polymerase and synthetic DNA templates provide an efficient method for producing small RNAs for biochemical and biophysical studies. The method is simple, scalable, and allows for the production of large quantities of homogeneous RNA. The study also highlights the advantages of this method over chemical synthesis, including the absence of side reactions that produce biologically inactive RNAs and the ability to amplify synthetic DNA up to 250-fold when converted to RNA. The study also shows that longer transcripts are produced more efficiently than shorter ones. The method is suitable for a wide range of RNA sequences and is particularly useful for producing RNAs with a variety of sequences near the 5' terminus. The study also shows that the T7 RNA polymerase can initiate transcription with different nucleotides at position +1, although the yield is reduced. The study concludes that the method is a reliable and efficient way to produce small RNAs for various applications.This study describes a method for synthesizing small, defined-length RNAs using T7 RNA polymerase and synthetic DNA templates containing the T7 promoter. The method allows for the production of RNA fragments ranging from 12 to 35 nucleotides in length, with milligram quantities achievable through optimized reaction conditions. The synthetic DNA templates, which are partially single-stranded and base-paired only in the promoter region, are as effective as linear plasmid DNA in transcription. Runoff transcripts initiate at a predictable position but may have one nucleotide more or less on the 3' end. The reactions also produce smaller oligoribonucleotides (2-6 nucleotides) as a result of abortive initiation events. Variants in the +1 to +6 region of the promoter can increase the variety of RNAs produced but reduce transcription efficiency. The study also shows that the non-template strand is not required for efficient transcription if the promoter region is double-stranded. The results indicate that the T7 RNA polymerase can add a non-template encoded nucleotide at the 3' end of the transcripts. The study further demonstrates that the length of the promoter does not significantly affect transcription efficiency, and that the T7 promoter is highly conserved from -17 to +6. The study also shows that the sequence beyond +2 has little effect on the yield of the reaction, while substitutions at +2 can reduce the yield. The study concludes that T7 RNA polymerase and synthetic DNA templates provide an efficient method for producing small RNAs for biochemical and biophysical studies. The method is simple, scalable, and allows for the production of large quantities of homogeneous RNA. The study also highlights the advantages of this method over chemical synthesis, including the absence of side reactions that produce biologically inactive RNAs and the ability to amplify synthetic DNA up to 250-fold when converted to RNA. The study also shows that longer transcripts are produced more efficiently than shorter ones. The method is suitable for a wide range of RNA sequences and is particularly useful for producing RNAs with a variety of sequences near the 5' terminus. The study also shows that the T7 RNA polymerase can initiate transcription with different nucleotides at position +1, although the yield is reduced. The study concludes that the method is a reliable and efficient way to produce small RNAs for various applications.