Binding properties: PNAs can form duplexes in either orientation, but the anti-parallel orientation is strongly preferred. This will be the orientation for all antisense and DNA probe type applications. The N-terminal of the PNA oligomer is equivalent to the 5¡¯-end of an oligonucleotide and is often referred to as ¡°the 5¡¯-end of the PNA¡±. A PNA/DNA-duplex will usually have a higher Tm than the corresponding DNA/DNA-duplex. As a rough rule, there will be an increase in Tm of about 1¡ÆC per base pair at 100 mM NaCl depending on the sequence.

Probe Length : Due to this higher affinity it is not necessary to prepare long PNA oligomers. For most applications an oligomer length of 12-18 is optimal. as opposed to the 25-40-mers, which is the typical length for an oligouncleotide probe. Bear in mind that the shorter a probe the more specific it is. The impact of a mismatch is greater, the shorter the sequence is. In many cases even shorter probes will work well, Longer PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize.

Purine Content : Purine rich PNA oligomers tend to aggregate, with G-rich oligomers being the worst. As a rule never have more than 7 purines in any stretch of 10 units. Observing this rule will dramatically reduce the likelihood that the PNA oligomer will aggregate. Then shorter the sequence the less attention needs to be paid to the sequence design.

Self-complementarity : Avoid self-complementary sequences such as inverse repeats, hairpin forming and palindromic sequences as PNA/PNA interactions are even stronger than PNA/DNA interactions. For example AATT would be OK but not CCGG or ATTATT. There is no problem with the synthesis but they are generally difficult to characterize and purify.