A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

No abstract

No abstract

Triple helix formation of oligodeoxynucleotides (ODNs) with a 15 base pair poly-purine DNA target in the HER2 promoter was examined by footprinting analysis. 7-deaza-2'-deoxyxanthosine (dzaX) was identified as a purine analogue of thymidine (T) which forms dzaX:A-T triplets. ODNs containing 2'-deoxyguanosine (G) and dzaX were found to form triple helices in an anti-parallel orientation, with respect to the poly-purine strand of the target DNA. In comparative studies under physiological K+ and Mg++ concentrations and at pH 7.2, the ODNs containing G and dzaX showed high affinity to the target sequence while the ODNs containing G and T were not able to bind. In the absence of added monovalent salts both ODNs showed high affinity to the target sequence. The substitution of 7-deaza-2'-deoxyguanosine for G substantially decreased the capacity of the ODNs to form triple helices under physiological conditions, indicating that dzaX may be unique in its ability to enhance triple helix formation in the anti-parallel motif.

The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure that contains four essential single-stranded nucleotides. Additional specificity is thought to be due to four or five ionic contacts between the protein and phosphates on the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5'-O-(1-thiotriphosphates) [NTP(alpha S)s], allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site were synthesized with different NTP(alpha S)s and tested for coat protein binding to deduce positions of thiophosphates that alter the binding affinity. Of the twenty-one phosphate positions in the molecule, two were found to decrease the Ka 3-fold when substituted with a thiophosphate, one position decreased the Ka 10-fold, and one position increased the Ka 10-fold. Substitution of any of the other 17 positions with thiophosphates does not alter the Ka. The four positions that alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates affect binding because they contact coat protein directly.