Amplification and re-generation of LNA-modified libraries

Area of Science:

• Molecular Biology
• Biochemistry
• Oligonucleotide Chemistry

Background:

• Locked nucleic acids (LNA) enhance oligonucleotide stability and nuclease resistance.
• Polymerases accepting LNA triphosphates enable LNA oligonucleotide library manipulation.
• In vitro selection requires robust oligonucleotide libraries for aptamer discovery.

Purpose of the Study:

• To propose and validate a scheme for amplifying and regenerating LNA-containing oligonucleotide libraries.
• To assess the feasibility of using LNA-modified libraries for in vitro selection of aptamers.
• To investigate the stability and sequence composition of LNA libraries over multiple selection rounds.

Main Methods:

• Maintenance of an oligonucleotide library with 40 randomized positions using LNA triphosphates (ATP, GTP, CTP, TTP).
• Seven rounds of mock in vitro selection without a target.
• Analysis of library sequence composition after rounds 1, 4, and 7, focusing on LNA monomer distribution.

Main Results:

• A decrease in LNA-A content from 20.5% (round 1) to 6.6% (round 7) was observed.
• A bias against successive LNA-A (poly-LNA adenosine tracts) and relative over-representation of single LNA-A were detected.
• Similar results were obtained when maintaining a library with LNA TTP, indicating general LNA monomer behavior.

Conclusions:

• Dispersed LNA monomers are tolerated within in vitro selection protocols.
• LNA-modified oligonucleotide libraries can be sustained for at least seven selection rounds, though LNA levels may decrease.
• The proposed scheme supports the discovery of native LNA aptamers and related oligonucleotide structures.