Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries

Abstract

The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.

Aptamer Bioinformatics

“…The binding capacity of G-quadruplex structures was exploited by McManus and Li, who integrated patterning into DNA libraries to select aptamers with quadruplex structures. They patterned the library with the following methods: inclusion of four G-tracts in the library while leaving the rest to be random; simplifying the complicated three-layer G-quadruplex into a two-layered structure; and four G 2 tracts were designed with three domains of random sequences for loop formation [ 71 , 78 ]. The authors first investigated the effective loop length for the folding of a quadruplex by adding three to seven thymidines between the G-tracts and characterizing by circular dichroism (CD).…”

“…( A ) Parallel quadruplex shows positive peaks at 265 and 295 nm; ( B ) antiparallel quadruplex with glycosidic bond angles of the same orientation shows bimodal spectra as positive peaks at 265 nm and 295 nm of the same intensity; ( C ) antiparallel quadruplex with glycosidic bond angles of opposite orientation—the CD shows a negative peak at 265 nm and a positive peak at 295 nm; ( D ) random library shows a peak at 280 nm but no peaks at 265 nm and 295 nm, indicating the absence of a quadruplex. Figure adapted from McManus and Li [ 78 ].…”

Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo

“…While canonical G4 DNA folds from a single DNA strand, G4 DNA can also form between two or even four separate strands of DNA [1]. More recently, the definition of G4-forming motifs has expanded to include those containing runs of two guanines, as well as spacers containing (many) more than seven nucleotides [2,3]. Given the high stability and wide range of potential G4 structures, is seems probable that mammalian cells have evolved different helicases with affinity for binding and unwinding of different G4 DNA structures.…”

G-quadruplex-based aptamers against protein targets in therapy and diagnostics

“…However, in some cases, even minor mutations in G4 sequences may have dramatic effects on aptamer stability and/or activity [147][148][149][150], inducing marked alterations in the G4 topology (e.g., an intramolecular antiparallel G4 can be converted into an intermolecular parallel one [151]) or causing the aptamer to target different epitopes of the protein [120]. Thus, the randomization process is always a compromise between the need for the highest sequence permutation and for a stable G4-folding [152]. In the case of an intermolecular scaffold, the combinatorial libraries usually contain several consecutive guanines (G-runs) with randomized flanks [98].…”