Guidelines for Sequence Design of PNA Oligomers

Specific Design Rules:

• Length: We will not synthesize a sequence with more than 18 bases, excluding linkers, amino acids and labels.
• Amino Acid Content: We are able to attach one Lysine or one Cysteine to either the N-terminus or the C-terminus.
• Purine Content: Purine-rich PNA oligomers tend to aggregate and have low solubility in aqueous solutions. To avoid aggregation, please follow the following specific guidelines:
  
  
1. Limit the purine content to 60%.

Example:
GAT TAG CAG TCT ACG (Acceptable - Purine content < 60%)

2. The maximum purine-stretch is 4 in a row. However, the maximum stretch of guanines is 3-in-a-row

Examples:
ATT AGG GGC ATC TAC (Not acceptable - 4 G's in a row)

CTA GAT AGA AGG TTC (Not Acceptable - 6 purines in a row)

3. Consider probing the complementary strand if you violate the above guidelines.

Examples:
GTA GAT GCC CCT AAT (Acceptable -Complement of the above sequence does not violate any of the guidelines)

GAA CCT TCT ATC TAG(Acceptable -Complement of the above sequence)

• Avoid self-complementary: sequences with inverse repeats, hairpins and palindromes. These types of probes are prone to aggregate, as PNA/PNA interactions are even stronger than PNA/DNA interactions.
  
  
1. Four base complements are acceptable, unless they contain only C and G. However CG complements are acceptable when interrupted by one or more bases.

Examples:
GAT AATT GCA (Acceptable - four bases complement)

GAT CCGG TAC (Not acceptable - CCGG complement)

TAT CCT GGT A (Acceptable CC, GG interrupted by a base)

2. Complements that are 6 bases or more in length are unacceptable.

Example:
CTA TTA ATG CA (Not acceptable - six bases complement)

3. Complements of 6 bases are acceptable when interrupted by one or more bases, unless only C and G are used.

Example:
AGT GCT ACT (Acceptable - interrupted six bases complement )

GCG GCT CGC (Not acceptable - G and C only complement

4. Complements of 8 bases are unacceptable even when interrupted by one or more bases.
   
   Example:
   ACTG T CAGT (Not acceptable - eight bases complement)

General Rules:

• We strongly recommend that you design anti-parallel custom PNA probes. PNAs can form duplexes in either orientation, but an anti-parallel orientation is strongly preferred and forms the most regular duplex. Anti-parallel is the preferred configuration for antisense and DNA probe-type applications. When the orientation of the PNA is anti-parallel, the N-terminal of the PNA probe is equivalent to the 5'-end of the DNA.
  
  
• PNA melting temperatures differ from those of DNA oligonucleotides. Because the PNA strand is uncharged, a PNA•DNA-duplex will have a higher T_m than the corresponding DNA-DNA-duplex. Typically there will be an increase in T_m of about 1°C per base pair at 100 mM NaCl. At lower salt concentrations the T_m differences will be even more dramatic. A 10-mer PNA will typically have a T_m of about 50°C, and a 15-mer PNA will typically have a T_m of 70° C.
  
  
• A PNA oligomer length of 12 to 17 units is optimal. Sequence length is primarily determined by the required specificity of the application. DNA applications that require more than 25 bases can be routinely performed with much shorter PNA probes. Long PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize. However, the shorter a sequence is, the more specific it is. Consequently, the impact of mismatch is greater for a short sequence.

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