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1.  G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold 
Nucleic Acids Research  2013;41(20):9524-9536.
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.
PMCID: PMC3814366  PMID: 23935071
2.  Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations 
PLoS ONE  2014;9(7):e102126.
Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.
PMCID: PMC4103837  PMID: 25036012
3.  Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin 
PLoS ONE  2012;7(7):e41702.
Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.
Principal Findings
Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.
We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.
PMCID: PMC3408503  PMID: 22860007
4.  Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers 
PLoS ONE  2012;7(4):e35990.
Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection.
In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition.
We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers
PMCID: PMC3338489  PMID: 22558297
5.  Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides 
Artificial DNA, PNA & XNA  2012;3(1):14-21.
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°Nm, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.
PMCID: PMC3368812  PMID: 22679529
enzymatic synthesis; modified nucleotide; polymerase; triphosphate; α-L-LNA

Results 1-5 (5)