DNA templates modified with C29-methoxyls at the last two nucleotides of the 59 termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single-and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
The EcoRI DNA dodecamer d(CGCGAATTCGCG)2 is investigated by state-of-the-art molecular dynamics
simulations. The BI → BII transition is shown to be a consequence of destacking processes of adjacent base
pairs and is coupled with migration of water from ionic phosphate to the sugar oxygen. Two-thirds of the
double-helical base steps temporarily are in the BII substate. The time-averaged BI/BII population ratio is
9.2, but at a given time, it can be as low as 2.7. Observed changes in the solvation properties closely match
recently reported spectroscopic features. The different time scales of all processes involved are discussed
with respect to experimental results. Formation of contact ion-pairs is demonstrated to be independent of
substate transitions.
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