Rakesh N. Veedu

Locked nucleic acids: promising nucleic acid analogs for therapeutic applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.

Locked nucleic acid as a novel class of therapeutic agents

Locked Nucleic Acid (LNA) is a nucleic acid analogue with unprecedented binding affinity and excellent specificity toward complementary RNA and DNA oligonucleotides. The remarkable properties of LNA have led to applications within various gene silencing strategies both in vitro and in vivo. In the present review, we highlight the uses of LNA for regulation of gene expression with emphasis on RNA targeting.

Enzymatic Incorporation of LNA Nucleotides into DNA Strands

Unlocking uses of locked nucleic acids: LNA nucleoside 5′-triphosphates have been synthesized, and their ability to serve as substrates for polymerases have been investigated. Phusion high-fidelity DNA polymerase was found to be an efficient enzyme for incorporating LNA nucleoside 5′-triphosphates into DNA strands.

Efficient enzymatic synthesis of LNA-modified DNA duplexes using KOD DNA polymerase.

Three different LNA-nucleoside triphosphates, LNA-TTP, LNA-ATP and LNA-5-methyl-CTP, were investigated as substrates for KOD DNA polymerase. The results reveal that KOD DNA polymerase is an efficient catalyst for template directed synthesis of DNA oligonucleotide duplexes containing a large number of LNA nucleotides by primer extension reactions. Furthermore, KOD DNA polymerase is shown to be suitable for the PCR amplification of an LNA-modified DNA duplex.

Enzymatic recognition of 2'-modified ribonucleoside 5'-triphosphates: towards the evolution of versatile aptamers.

The quest for effective, selective and nontoxic nucleic‐acid‐based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target‐validation studies and therapeutic applications involving antisense, RNAi, aptamer, and ribozyme‐based technologies. Since their invention in the early 1990s, aptamers have emerged as a very promising class of therapeutics, with one drug entering the market for the treatment of age‐related macular degeneration. To combat the limitations of aptamers containing naturally occurring nucleotides, chemically modified nucleotides have to be used. In order to apply modified nucleotides in aptamer drug development, their enzyme‐recognition capabilities must be understood. For this purpose, several modified nucleoside 5′‐triphosphates were synthesized and investigated as substrates for various enzymes. Herein, we review studies on the enzyme‐recognition of various 2′‐sugar‐modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers.

Locked nucleic acid nucleoside triphosphates and polymerases: on the way towards evolution of LNA aptamers

Among numerous nucleic acid analogs reported in the past decades, locked nucleic acid (LNA) has received substantial attention and has become a significant tool within chemical biology disciplines like molecular biology research, diagnostics and therapeutic development. However, despite their obvious structurally unique properties, LNA-based aptamers for diagnostic and therapeutic applications remain largely unexplored. Future evolution of LNA oligonucleotide aptamers will depend on scientific breakthroughs relating to enzymatic polymerization using LNA nucleoside triphosphates as substrates. Herein, we highlight recent developments in this direction using various polymerases.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K+ rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5′ with 3 nt and 3′ with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

Multifunctional and multitargeted nanoparticles for drug delivery to overcome barriers of drug resistance in human cancers

The recurrence and metastatic spread of cancer are major drawbacks in cancer treatment. Although chemotherapy is one of the most effective methods for the treatment of metastatic cancers, it is nonspecific and causes significant toxic damage. The development of drug resistance to chemotherapeutic agents through various mechanisms also limits their therapeutic potential. However, as we discuss here, the use of nanodelivery systems that are a combination of diagnostics and therapeutics (theranostics) is as relatively novel concept in the treatment of cancer. Such systems are likely to improve the therapeutic benefits of encapsulated drugs and can transit to the desired site, maintaining their pharmaceutical properties. The specific targeting of malignant cells using multifunctional nanoparticles exploits theranostics as an improved agent for delivering anticancer drugs and as a new solution for overriding drug resistance.

In vitro incorporation of LNA nucleotides

An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product.