Enzymatic synthesis of modified DNA by PCR

CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase

“…16 In contrast, replication of nucleotide analogues functionalized with smaller substituents appears comparatively straightforward. [53][54][55][56][57] The most advanced of these systems was described by Ja ¨ger and Famulok, who, through rigorous optimization of reaction conditions, achieved successful PCR amplification of short (<100bp) fragments in which all nucleotide analogues were functionalized with a range of small nonfluorescent groups. 35,58 We substituted only one base (dC) but with a large, heterocyclic, charged cyanine dye substituent (Cy3-, Cy5-dC), which turned out to be a challenging substrate at full substitution.…”

“…A further challenge is presented by the replication of such polymers as the bulky substituents are now also present in the template strand, which can cause additional steric strain resulting in pausing or abortive synthesis . In contrast, replication of nucleotide analogues functionalized with smaller substituents appears comparatively straightforward. − The most advanced of these systems was described by Jäger and Famulok, who, through rigorous optimization of reaction conditions, achieved successful PCR amplification of short (<100bp) fragments in which all nucleotide analogues were functionalized with a range of small nonfluorescent groups. , We substituted only one base (dC) but with a large, heterocyclic, charged cyanine dye substituent (Cy3-, Cy5-dC), which turned out to be a challenging substrate at full substitution. Nevertheless, two rounds of directed evolution of polymerase function by spCSR, targeting the conserved A- and C-motif regions in the polymerase active site, yielded E10 (Pfuexo-: V93Q, V337I, E399D, N400D, R407I, Y546H), a variant polymerase with a substantially enhanced capacity to utilize Cy3- and Cy5-dCTP as substrates.…”

Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy

“…The modification of the 2‘-hydroxyl group of the sugar ribose is straightforward and sufficient to enhance the stability of a RNA molecule in vivo and in living organisms. Other potential sites for chemical modification that may be beneficial for the properties of therapeutic aptamers are the purine and pyrimidine residues, − sugars, − and phosphate residues, which all have been shown to be compatible with template-directed enzymatic incorporation into DNA and with all necessary steps required for a complete in vitro selection cycle. , In general, modified nucleotides are increasingly being utilized in all categories of therapeutic oligonucleotides to increase nuclease resistance, target affinity, and specificity . As summarized in Figure , ‘-amino, ,− 2‘-fluoro, ,,, 2‘-methoxy, , and 4‘-thio 180 groups have been used directly in SELEX experiments to increase the stability of aptamers.…”