Ligation reactions with catalipid release
Experiments were carried out to investigate ligation reaction using
activated oligonucleotide-catalipid conjugate 21 (Figure21): To 10 nmol
5'-amino-oligonucleotide NH2-CGGAA (15) and 4 nmol 5'-Cy3
labeled template molecule Cy3-TTCCGCGGAA (16) a volume of
10 µl of a 1 mM solution of 21 in ligation buffer (0.1M HEPES, pH
7.2, 50 mM NaCl, 20% acetonitrile) was added. The reaction mixture was
incubated for several hours at 20°C and ligation product formation
of Cy5-TTCCGpnCGGAA (17) was analyzed by RP-HPLC (Figure
22).
Figure 22 shows that Cy5 labeled reaction components (14, 17 and 21)
can be selectively detected at a wave length of 644 nm. Samples were
taken after a reaction time of 20 minutes, 1, 2, 3, 4, 6, 24, 34 hours
and subsequently analyzed by RP-HPLC.
The ligation product Cy5-TTCCGpnCGGAA (17) has a retention time of 9 minutes (A), the peak at 10 minutes (B) refers to the hydrolyzed precursor 21 (Cy5-TTCCGp 14) and the peak at a retention time of 13 minutes (C) to the activated oligonucleotide-catalipid conjugate 21. The ligation product 17 could also be identified by means of MALDI-TOF mass spectrometry (Figure 23).
Ligation reactions using reactive oligonucleotide-catalipid
conjugates based on catalipids MC6H (18), MC10H
(20) show similar behavior.
Investigations towards reaction kinetics of the catalipid-activated ligation reaction are in progress. To gain a more quantitative understanding into the topic of replication coupled to metabolism and compartmentation, model experiments were performed based on the concept described in section 3. It was not possible to give final evidence of phosphorimidazolide formation on the outer side of giant vesicles by means of (light) microscopy. NMR spectroscopy was employed therefore.
Figure 21
Figure 22
Figure 23