Agarose gel electrophoresis of various plasmids was carried out with and without PNA binding and restriction digestion. A: Supercoiled (SC) DNA substrates were converted to linear (L) upon cleavage by restriction digestion. In pFR(3/4) and pFR(1/2), the Hind III and EcoRI sites, respectively, are partially within the PNA binding sequence, thus HindIII and EcoRI were used for pFR(3/4) and pFR(1/2), respectively. For the vector plasmid pBluescript SK(+), both HindIII (lanes 9 and 11) and EcoRI (lanes 13, 15) demonstrate the absence of non-specific PNA effect on these enzymes. It can be seen that PNA protected the majority of its targets from cleavage (lanes 3 and 5). B: Similar experiments with linear DNA substrates.

Agarose gel electrophoresis of plasmids was carried out with and without the (GAA/CTT)₃₉ inset, and with or without PNA binding at various concentrations of NaCl.
A: relaxed DNA; B: negatively supercoiled DNA.

Autoradiograph of the transcription products separated in denaturing acrylamide gel. A pronounced effect was evident only when preincubation with PNA preceded the addition of salt (lanes 1 and 5). Gel-shifts of linear DNA on the agarose gel upon PNA binding (at the bottom of the Figure) confirmed that PNA binds DNA only when 200 mM NaCl were added after preincubation with PNA.

A: Substrates with and without T7 promoter. Only PNA-bound substrates are shown. Promoter-independent transcription (shown by three-legged arrows) in principle could start from any point within single-stranded regions, but it might have some preference for the single-strand/duplex junction. Once it starts, it would continue to the end of the substrate.
B: Transcription from substrates shown in A. Lane 7 corresponds to Substrate I, lane 9 corresponds to Substrate II, lane 3 corresponds to Substrate III, lane 5 corresponds to Substrate IV. The designation “n/r” (“no repeats) indicates the vector plasmid pBluescript SK(+). Top panel: a lower exposure of the top area of the same gel containing run-off products is shown.

A: PNA-DNA hybrid. PNA bases are shown in bold. PNA binds the homopurine DNA strand, displacing the homopyrimidine DNA strand. The bottom PNA strand is bound to DNA via Watson-Crick hydrogen bonds, and the top PNA strand is bound to DNA via Hoogsteen hydrogen bonds. In the top strand, cytosines are replaced by pseudoisocytosines (J) which, in contrast to cytosines, do not require protonation for Hoogsteen base pairing. The top and the bottom PNA strands are connected by a flexible linker of three residues of 8-amino-3,6-dioxaoctanoic acid. B: Typical DNA substrate used in the transcription experiments. The PNA binding insert was cloned in each of the two possible orientations.

Agarose gel electrophoresis of relaxed plasmids was carried out with and without the (GAA/CTT)₃₉ insert, and with or without PNA binding. The appearance of the faster-migrating, positively supercoiled plasmid is apparent in lane 1.

The autoradiograph shows the transcription products resolved by denaturing acrylamide gel electrophoresis. The designation “n/r” (“no repeats”) indicates the vector plasmid pBluescript SK(+). L-100 and L-10 are 100 nt and 10 nt denatured DNA ladders, respectively. The positions of the inserts are shown at right. A: Linear DNA substrates. B: Negatively supercoiled DNA substrates.

A: The substrate used for transcription. Single-stranded circular DNA corresponding to the template strand of pBlue-iCTT39 plasmid was annealed to the promoter oligo (PO) and to the restriction oligo (RO) that contains the corresponding restriction site that allows linearization of the template. The first 17 nucleotides of the promoter oligo for pBlue-iCTT plasmid correspond to the top strand of the T7 promoter (symbolized by the gray arrow), the rest of the oligo (54 nucleotides) covers the area from the promoter to the CTT insert. A similar promoter oligo was used for the vector plasmid pBluescriptSK(+), except that it covered 48 nucleotides of the downstream area instead of 54. The distance from the CTT-insert to the EcoRI cleavage site was 21 nt, thus the expected run-off in this case is around 0.19 kb. The expected run-off for the pBlue-iCTT39 plasmid and the AflIII oligo is 0.63 kb. The expected run-of for the pBluescript SK(+) plasmid and the AflIII oligo is 0.51 kb.
B: Transcription products from the substrates described in A with and without Klenow extension, separated in a denaturing acrylamide gel. CTT stands for pBlue-iCTT39 plasmid, BS stands for pBluescript SK(+) plasmid. The positions of corresponding run-off products were consistent with expectation within the gel resolution. Lane 1 corresponds to transcription in the absence of DNA substrates, which did not produce any transcript in our conditions, although in principle such reaction is possible [52]. Lane 2 shows transcripts from the promoter oligo alone, which were also present in the rest of the lanes because of excess of the unbound promoter oligo. Although the exact nature of these products is not known, this kind of reaction was described before and it did not interfere with our analysis. When the samples were treated with Klenow before transcription, these products became shorter most likely because of partial digestion of free promoter oligo by Klenow.