PNA strands were synthesized in-house using solid phase peptide synthesis. Lysine residues were attached at either end of the PNA sequence to improve solubility. Fmoc-PNA monomers (PNA-Bio, Inc.) were coupled to a low loading Tentagel-S-RAM resin using 4 eq. PNA, 3.95 eq. PyBOP, and 6 eq. diisopropylethylamine (DIEA). Lysine and glycine residues were reacted in the same way. Following each coupling, the peptide was deprotected in 20% piperidine in DMF. N-maleoyl-β-alanine (Sigma) was coupled to the N-terminus under the same coupling conditions. The peptide was then cleaved from the resin in 95% trifluoroacetic acid (TFA), 2.5% H2O, and 2.5% triisopropylsilane. The peptide was dissolved in aqueous solution with 0.1% TFA, filtered, and purified by HPLC using a C-18 Gemini column (Phenomenex) with a mobile phase of acetonitrile containing 0.1% TFA. Purity of the PNA products was analyzed with MALDI-TOF mass spectrometry on a Bruker Daltonics microflex. The sequence of the synthesized PNA strand is: (Maleimide)-GGK-cagtccagt-K-(CONH2), and the complementary ssDNA is: 5’-Oligo-TT-ACTGGACTG-3’ (melting temperature predicted: 56.7°C). The sequence has been designed to have a melting temperature above 55°C (predicted with the PNA tool: https://www.pnabio.com/support/PNA_Tool.htm , from PNA Bio, Inc.) and orthogonal to the sequence of M13mp18 and validated using NCBI BLAST online tool.
Daltonics microflex
Manufactured by Bruker
The Daltonics microflex is a compact and versatile matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. It is designed for the analysis of a wide range of samples, including proteins, peptides, and small molecules. The microflex provides high-performance analytical capabilities in a benchtop instrument format.
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2 protocols using daltonics microflex
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Synthesis and Characterization of PNA-DNA Hybrid
2
Synthesis and Characterization of PNA-DNA Hybrid
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PNA strands were synthesized in-house using solid phase peptide synthesis. Lysine residues were attached at either end of the PNA sequence to improve solubility. Fmoc-PNA monomers (PNA-Bio, Inc.) were coupled to a low loading Tentagel-S-RAM resin using 4 eq. PNA, 3.95 eq. PyBOP, and 6 eq. diisopropylethylamine (DIEA). Lysine and glycine residues were reacted in the same way. Following each coupling, the peptide was deprotected in 20% piperidine in DMF. N-maleoyl-β-alanine (Sigma) was coupled to the N-terminus under the same coupling conditions. The peptide was then cleaved from the resin in 95% trifluoroacetic acid (TFA), 2.5% H2O, and 2.5% triisopropylsilane. The peptide was dissolved in aqueous solution with 0.1% TFA, filtered, and purified by HPLC using a C-18 Gemini column (Phenomenex) with a mobile phase of acetonitrile containing 0.1% TFA. Purity of the PNA products was analyzed with MALDI-TOF mass spectrometry on a Bruker Daltonics microflex. The sequence of the synthesized PNA strand is: (Maleimide)-GGK-cagtccagt-K-(CONH2), and the complementary ssDNA is: 5’-Oligo-TT-ACTGGACTG-3’ (melting temperature predicted: 56.7°C). The sequence has been designed to have a melting temperature above 55°C (predicted with the PNA tool: https://www.pnabio.com/support/PNA_Tool.htm , from PNA Bio, Inc.) and orthogonal to the sequence of M13mp18 and validated using NCBI BLAST online tool.
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