# When the Position of Pendant Groups Makes the Difference in G‑Quadruplex Behavior: The Case of Bis-Conjugated Thrombin-Binding Aptamers

## TL;DR

This paper shows how changing the position of chemical groups on a DNA sequence can significantly affect its stability, resistance to enzymes, and ability to block blood clotting.

## Contribution

The study reveals that the position of pendant groups on thrombin-binding aptamers affects their G-quadruplex behavior and anticoagulant activity.

## Key findings

- N-TBA-p showed higher thermal stability and nuclease resistance compared to p-TBA-N and TBA.
- N-TBA-p exhibited greater anticoagulant activity due to improved thrombin recognition.
- Molecular dynamics analyses explained the structural basis for the observed differences in behavior.

## Abstract

In the search for effective and low-toxicity anticoagulant
agents,
the G-quadruplex-forming thrombin-binding aptamer (TBA) with sequence
5′-GGTTGGTGTGGTTGG-3′, able to selectively recognize
the fibrinogen-binding exosite I of the thrombin enzyme, emerged as
a promising therapeutic and surgical tool. In this frame, we recently
synthesized and evaluated a library of TBA analogues carrying a naphthalene
diimide (N) moiety and a 3-hydroxypropylphosphate (p) either at the 5′- or 3′-end of the TBA sequence.
Interestingly, N-TBA-p and p-TBA-N analogues,
having the same pendant groups at 5′- or 3′-end but
in reversed position, showed very different behavior in terms of thermal
stability, nuclease resistance in serum, and anticoagulant activity. N-TBA-p showed enhanced properties compared to both p-TBA-N and the parent TBA and thus emerged as a very promising
candidate for future in vivo studies. Here, by in-depth molecular
dynamics-based analyses, we disclosed the structural features determining
the higher thermal stability and nuclease resistance as well as the
higher anticoagulant activity due to thrombin recognition, experimentally
observed for N-TBA-p than p-TBA-N and TBA.