Effect of Loop Sequence and Size on DNA Aptamer Stability

Abstract

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

“…2. [14][15][16]21] in the presence of potassium ions. Such CD pattern is observed when antiparallel G-quadruplex is formed.…”

Specific loop modifications of the thrombin‐binding aptamer trigger the formation of parallel structures

“…The loops connecting G-quartets have a strong influence on the folding and stability of intramolecular quadruplexes. Different loop lengths and sequences can either stabilize or destabilize the G-quadruplex [23][24][25][26][27][28]. For example, reduction of the two lateral TT loops of TBA to a single thymidine caused complete disruption of the quadruplex structure, whereas extension to TTT did not alter the stability [23].…”

“…Different loop lengths and sequences can either stabilize or destabilize the G-quadruplex [23][24][25][26][27][28]. For example, reduction of the two lateral TT loops of TBA to a single thymidine caused complete disruption of the quadruplex structure, whereas extension to TTT did not alter the stability [23]. Significant changes in stability have also been found when thymidines in the lateral loops have been changed to other coding nucleotides [29,30], or to derivatives with modified backbones [31][32][33][34][35].…”

Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates

“…[8][9][10][11][12][13][14][15][16][17][18] Among these sensing technologies, electrochemical aptasensors have attracted particular attention because they provide a simple, sensitive, and selective platform for thrombin detection. A SPR spectroscopic aptasensor allows use of a microsecond time-resolved change of SPR reflectance.…”