Chemical Methods of DNA and RNA Fluorescent Labeling

Abstract

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.

Efficient RNA 5′-adenylation by T4 DNA ligase to facilitate practical applications

“…1), RNA 59-adenylation using T4 DNA ligase and a-32 P-ATP is an alternative approach to RNA 59-radiolabeling that may be of practical utility. Finally, because the adenylate moiety has an ribose 29,39-diol group that may be oxidized with sodium periodate to aldehydes, RNA 59-adenylation enables labeling of the 59-terminus by subsequent reductive amination or other coupling reactions using a variety of amine reagents combined with biophysical probes (Wells and Cantor 1977;Odom et al 1980;Proudnikov and Mirzabekov 1996). This overall 59-labeling approach will be particularly useful when a method for RNA labeling is sought that does not require incorporation by solid-phase synthesis of special 59-terminal RNA nucleotides and the RNA 39-terminus cannot be used as a modification site, e.g., when it is blocked as a 29,39-cyclic phosphate.…”

Introduction to Microarray‐Based Detection Methods

“…Several methods have been suggested to alleviate this problem, such as the use of helper oligonucleotides [130] and a two-probe proximal chaperon detection system [158] to mitigate the effects of target secondary structure hindrances, an appropriate labeling method [63], and a protocol to achieve optimized target lengths [126,164,197]. Since long targets can form secondary and tertiary structures that hinder efficient probe-target duplex formation, the sizes of the target molecule and its amplicon are often reduced via chemical, enzymatic or thermal fragmentation methods [24,87,104,126,138,158]. Liu et al have recently elegantly reviewed these issues and experimentally demonstrated that microarray hybridizations with short rRNA fragments were more dependent on target sequence than on the competition between probe-target interaction and RNA self-folding [103].…”

“…Various enzyme-assisted hybridization strategies (also used in SNP and resequencing assays [52,98]) are being applied because of their promise in strongly discriminating single mismatches located near the 3 0 -end of microarray probes [34,75,101,119,135,138,145].…”

A general two-step strategy to synthesize lariat RNAs

“…Cyclic phosphate termini are also readily removed after the reaction if a free 2¢,3¢-diol is desired (Schürer et al 2002). As an alternative to capping with a 2¢,3¢-cyclic phosphate, the 3¢-terminal ribose moiety of the substrate could be oxidized with sodium periodate (Proudnikov and Mirzabekov 1996). Such periodate oxidation would be an acceptable capping approach if the resulting 3¢-terminal aldehyde functional groups do not interfere with subsequent applications of the RNA.…”