The faithful recognition of the interstrand hydrogen bonds between complementary nucleobases forms the foundation of the genetic code. The ability to replicate DNA containing a stable third base pair would allow for an expansion of the information content of DNA by supplementing the existing two base pairs of the genetic alphabet with a third. We report the optimization of unnatural nucleobases whose pairing in duplex DNA is based on interbase hydrophobic interactions. We show that the stability and selectivity of such unnatural base pairs may be comparable to, or even exceed, that of native pairs. We also demonstrate that several unnatural base pairs are incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase I with an efficiency equivalent to that of native DNA synthesis. Moreover, the unnatural bases are orthogonal to the native bases, with correct pairing being favored by at least an order of magnitude relative to mispairing.
Six novel unnatural nucleobases have been characterized that form stable base pairs in duplex DNA,
relying not on hydrogen bonds, but rather on interbase hydrophobic interactions. These nucleobases are
derivatives of the hydrophobic base pair between 7-azaindole (7AI) and isocarbostyril (ICS). Derivatives of
7AI and ICS were examined that have increased hydrophobic surface area, as well as increased polarizability.
As observed with 7AI and ICS, these derivatives are recognized as substrates by Klenow fragment of Escherichia
coli DNA polymerase I. The unnatural base pair between pyrrolopyrizine (PP) and C3-methylisocarbostyril
(MICS) is enzymatically incorporated into DNA with high efficiency (k
cat/K
M = 106 M-1 min-1) and moderate
selectivity. These studies represent a significant step toward the generation of a stable, orthogonal base pair
that can be enzymatically incorporated into DNA with good fidelity.
Selective
immunoproteasome inhibition is a promising approach for treating
autoimmune disorders, but optimal proteolytic active site subunit
inhibition profiles remain unknown. We reveal here our design of peptide
epoxyketone-based selective low molecular mass polypeptide-7 (LMP7)
and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit
inhibitors. Utilizing these and our previously disclosed low molecular
mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement
of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent
cytokine expression inhibition and in vivo efficacy in an inflammatory
disease model. These and additional findings toward optimized solubility
led the design and selection of KZR-616 disclosed here and presently
in clinical trials for treatment of rheumatic disease.
Relying on only interbase hydrophobic packing for bonding, several unnatural nucleobases similar to 1 can form selective and stable self‐pairs in duplex DNA. These unnatural nucleobases should make it possible to increase the stringency and information content of hybridization or encoding experiments.
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