Elena A. Lesnik

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Remarkable enhancement of binding affinity of Heterocycle-modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2-imidazolylpropylguanine and N2-imidazolylpropyl-2-aminoadenine modified oligonucleotides

Abstract Oligonucleotides containing novel N2-Imidazolylpropylguanine and N2-Imidazolylpropyl-2-aminoadenine moieties were synthesized and studied for their hybridization and biophysical properties. Interestingly, these heterocycle modified oligonucleotides showed a remarkable enhancement of heteroduplex binding affinity when hybridized to complementary DNA.

Zwitterionic oligonucleotides with 2′-O-[3-(N,N-dimethylamino)propyl]-RNA modification: synthesis and properties

Abstract A novel 2′-modification, 2′-O-[3-(N,N-dimethylamino)propyl] or 2′-O-DMAP, has been incorporated into oligonucleotides and compared to the known 2′-O-(3-aminopropyl) or 2′-O-AP modification for antisense properties. The 2′-O-DMAP modified oligonucleotides exhibit very high nuclease resistance like the 2′-O-AP modification due to the ‘charge effect’ and maintain high binding affinity to target RNA relative to known modifications when a few 2′-O-DMAP residues are dispersed throughout the oligonucleotide.

2′-O-Carbamate-containing oligonucleotides: synthesis and properties

Abstract In order to evaluate the effect of a new 2′-carbohydrate modification on the hybridization properties of oligonucleotides, uridine 2′- O -carbamates were synthesized and incorporated into DNA strands. The key intermediate in the synthesis, a mixed succinimide carbonate 2 , was treated with various amines to give 2′- O -carbamates 3 . Thermal melting studies of modified oligonucleotides revealed that the presence of the 2′- O -carbamate modification significantly destabilized DNA/RNA duplexes. A molecular-modeling study indicated that unfavorable steric interactions between the hydrogen of the NH group from the carbamate substituent and the anomeric hydrogen of the sugar residue on the same strand of the duplex may be the contributing factor causing destabilization.

Triplex Formation Between DNA and Mixed Purine-Pyrimidine PNA Analog with Lysines in Backbone

Abstract Melting UV experiments and mixing curves indicated slow triplex formation between lysine comprising PNA and DNA complement in 100mM Na+ solution.

Improving antisense oligonucleotide binding to human serum albumin: Dramatic effect of ibuprofen conjugation

The interaction of antisense oligonucleotides with serum and cellular proteins determines their pharmacokinetic (transport to and distribution in target tissues) and pharmacodynamic (binding to the mRNA target) properties and hence their eventual pharmacology. In general, binding of drugs to serum albumin, 2-macroglobulin, immunoglobulins, and lipoproteins in the bloodstream governs their transport and tissue distribution. The first generation antisense compounds, 2 -deoxyphosphorothioate oligonucleotides (containing P S linkages, where R H and X S in Scheme 1), bind rapidly to serum and cellular

Synthesis and Incorporation of 2′-O-Methyl-Pseudouridine into Oligonucleotides

Abstract A short multigram synthesis of 2′-O-methylpseudouridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogens. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2′-O-methyluridine.

Synthesis of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified oligonucleotides: hybridization affinity, resistance to nuclease, and protein binding characteristics

Abstract Synthesis of a series of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties.

Positioning of Functionalities in a Heteroduplex Major Groove: Synthesis of 7-Deaza-2-Amino-2′-deoxyadenosines

Abstract We have synthesized the 7-iodo-, 7-cyano-and 7-propynyl-7-deaza-2-ainino-2′-deoxyadenosines and incorporated each into several oligonucleotide (ODN) sequences. These oligonucleotides exhibit enhanced binding affinities to RNA complements relative to unmodified sequences.