Glycosaminoglycans (GAG) are a family of large, linear, sulfated polysaccharides produced in mammals and
other organisms. GAGs play diverse roles in tissue development/growth, inflammation, blood coagulation, viral
infection, and amyloid plaque formation. As a result, GAGs have been used as biomarkers for many diseases.
They are also the most widely used anticoagulant in medicine. Because of their biological activities, interest in
structure-activity relationships of GAGs has always been high. However, due to their size, complexity and het-
erogeneity, analysis of GAG structures using conventional ensemble techniques has always been challenging.
There is currently no method to sequence these important polysaccharides. We have been exploring single-
molecule techniques for determining GAG structures for several years. In this proposal, we want to explore the
possibility of using fluctuations in the electrical conductance of GAG lyases to elucidate the structures of GAGs.
This idea originates from our work on single protein conductance measurements that showed many non-redox
active proteins can conduct electricity. In addition, the conductance of proteins is often sensitive to conformation
dynamics triggered by substrate binding or catalytic activity, allowing them to act as single-molecule sensors for
substrates. We have applied such measurements to DNA polymerases and showed current fluctuations in the
polymerase correlated with enzyme conformation changes during DNA replication. The generalization of this
idea potentially allows any biopolymer to be sequenced as long as a processive metabolizing enzyme can be
found for the polymer. Such enzymes were usually scarce for GAGs. However, a new class of processive exolytic
bacterial GAG lyases that degrade GAGs from their reducing end has just been identified. In this proposal, we
want to apply this technique to this class of enzymes to determine whether fluctuations in the conductance of
these lyases are reflective of the structures of the substrates being processed. Because such a method requires
no homogeneous samples, can sequence longer GAG polymers, and can provide high-resolution information,
we think its realization will be a dramatic improvement over all existing techniques. In particular, we want to
complete the following two aims: 1) Leveraging the technologies we developed to connect DNA polymerases to
electrodes, we will design and produce lyases that can be attached to electrodes specifically and optimize the
anchoring points to maximize conductance and sensitivity to substrate binding while retaining the enzyme activity.
2) We will prepare a library of structurally defined GAG ligands and probe the enzymes with the ligands to de-
termine if the substrate-induced fluctuations in the enzymes’ conductance contain information that can be used
to identify the structures of the substrates. Completion of these aims will provide the crucial foundation for real-
izing the goal of developing a general method for sequencing GAGs.