Systematic study of constraints imposed by modified nucleoside triphosphates with protein-like side chains for use in in vitro selection

Abstract

Successful selection of modified DNAzymes depends on the potential for modified nucleoside triphosphates (dNTPs) to replace their unmodified counterparts in enzyme catalyzed primer extension reactions and, once incorporated, to serve as template bases for information transfer prior to PCR amplification. To date, the most densely modified DNAzymes have been selected from three modified dNTPs: 8-histaminyl-deoxyadenosine (dATP), 5-guanidinoallyl-deoxyuridine (dUTP), and 5-aminoallyl-deoxycytidine (dCTP) to provide several RNA-cleaving DNAzymes with greatly enhanced rate constants compared to unmodified counterparts. Here we report biophysical and enzymatic properties of these three modified nucleosides in the context of specific oligonucleotide sequences to understand how these three modified nucleobases function in combinatorial selection. The base-pairing abilities of oligonucleotides bearing one or three modified nucleosides were investigated by thermal denaturation studies and as templates for enzymatic polymerization with both modified and unmodified dNTPs. While we address certain shortcomings in the use of modified dNTPs, we also provide key evidence of faithful incorporation and enzymatic read-out, which strongly supports their continued use in in vitro selection.

Enzymatic Synthesis, Amplification, and Application of DNA with a Functionalized Backbone

“…Theu tility of DNAf or these applications stems from its precisely controllable sequence composition and base pairing, which enables the polymerase chain reaction (PCR) amplification of specific polymers and/or the control of their assembly into higher order structures;h owever their physicochemical properties are limited to those possessed by the four nucleobases,s ugar, and phosphate.T hus,t ot ake full advantage of the precise sequence composition available with DNA, modifications have been sought that may be introduced to one or more of the triphosphates without interfering with their polymerase recognition. Themost success has been achieved with nucleobase modifications, [11][12][13][14][15][16][17][18] which have been used to develop aptamers with protein-like properties, [19,20] or to introduce sites for the attachment of other moieties by click chemistry. [21][22][23][24][25] However this approach is mostly limited to pyrimidine modifications,and the modified oligonucleotides themselves are not well recognized by polymerases,p reventing their production via PCR-mediated amplification.…”

Modulation of BACE1 Activity by Chemically Modified Aptamers

“…Despite the fact that 5‐CldUTP residues were described to pair with dATP but also with dGTP, we observed no significant misincorporation in place of 5‐CldUTP residues (Section S7). This is consistent with a recent work in which the base‐pairing abilities of oligonucleotides incorporating one or three modified nucleosides were investigated and showed correct incorporation of modified dNTPs …”

A densely modified M2+-independent DNAzyme that cleaves RNA efficiently with multiple catalytic turnover

“…Previously, we showed that the dC aa s greatly stabilize complementary interactions, which we anticipated would constitute an advantage in terms of substrate recognition and would offset the slightly destabilizing effects of the dA im . 72 Nevertheless, we also recognized that these modifications would limit the ability to alter the 3′-guide arm sequence. To test this, we replaced the dA im in the guide arm with dG; the resulting DNAzyme had limited yet detectable activity against the complementary substrate (∼20%), and very little activity against the original substrate that would contain a G-U wobble mismatch (see Fig.…”

“…Recently, we demonstrated that Sequenase, the enzyme used to prepare Dz7-38-32, inserts 1–3 with high fidelity while Taq recopies the resulting modified strands with equally high fidelity resulting in less than 1% mutation overall. 72 That insertion and read-out result in the selection of a given DNAzyme containing 1–3 , reflects the incontrovertible consequences of semiconservative replication along with Chargaff's rules, which ensure that a modified nucleoside will either be perpetuated through rounds of selection or otherwise irretrievably lost. More importantly, we demonstrated that the enzymatic synthesis of these DNAzymes provides homogeneous species that are as pure as any other enzymatically synthesized DNAzyme or ribozyme.…”