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The Effect of Nucleic Acid Modifications on Digestion by DNA Exonucleases by Greg Lohman, Ph.D., New England Biolabs, Inc

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The Effect of Nucleic Acid Modifications on Digestion by DNA Exonucleases by Greg Lohman, Ph.D., New England Biolabs, Inc be INSPIRED FEATURE ARTICLE drive DISCOVERY stay GENUINE The effect of nucleic acid modifications on digestion by DNA exonucleases by Greg Lohman, Ph.D., New England Biolabs, Inc. New England Biolabs offers a wide variety of exonucleases with a range of nucleotide structure specificity. Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5´ end and/or the 3´ end of polynucleotides, and can also act on RNA. Exonucleases have many applications in molecular biology, including removal of PCR primers, cleanup of plasmid DNA and production of ssDNA from dsDNA. In this article, we explore the activity of commercially available exonucleases on oligonucleotides that have chemical modifications added during phosphoramidite synthesis, including phosphorothioate diester bonds, 2´-modified riboses, modified bases, and 5´ and 3´ end modifications. We discuss how modifications can be used to selectively protect some polynucleotides from digestion in vitro, and which modifications will be cleaved like natural DNA. This information can be helpful for designing primers that are stable to exonucleases, protecting specific strands of DNA, and preparing oligonucleotides with modifications that will be resistant to rapid cleavage by common exonuclease activities. The ability of nucleases to hydrolyze phosphodi- of exonucleases available from NEB can be found Figure 1: ester bonds in nucleic acids is among the earliest in our selection chart, Common Applications of Examples of exonuclease directionality nucleic acid enzyme activities to be characterized Exonucleases and Non-specific Endonucleases, at → (1-6). Endonucleases cleave internal phosphodiester go.neb.com/ExosEndos. 3´ 5´ exonuclease bonds, while exonucleases, the focus of this article, What about cases where you only want to degrade 5´ 3´ must begin at the 5´ or 3´ end of a nucleic acid some of the ssDNA in a reaction? Or, when you 3´ 5´ strand and cleave the bonds sequentially (Figure 1). want to make ssDNA from a dsDNA substrate, but Exonucleases may be DNA or RNA specific, and which strand is degraded matters greatly? What can act on single-stranded or double-stranded 5´→3´ exonuclease about cases where the ends of your nucleic acids nucleic acids, or both. Double-strand specific are modified—will exonucleases still digest the sub- exonucleases may initiate at blunt ends, nicks, or 5´ 3´ strate, or cleave the modification? Several methods short single-stranded 5´ or 3´ overhangs, though 3´ 5´ depend on selective protection of polynucleotides, most exonucleases are active on a subset of these such as protection of primers from degradation by structures. For a summary of the substrate specificity polymerase exonuclease domains (17), selective Bidirectional exonuclease of exonucleases available from NEB, view our protection of one strand of a DNA duplex for the selection chart, Properties of Exonucleases and 5´ 3´ production of ssDNA (14-16), and the protection Non-specific Endonucleases, at go.neb.com/ of polynucleotides from degradation by serum 3´ 5´ ExosEndos. nucleases, as in the case of RNA interference A variety of DNA exonucleases have been char- drugs (18, 19). In each of these cases, it is critical Pictured are double stranded exonucleases with a 3´ to acterized from many different organisms;in vivo, to understand the influence of modifications on 5´ polarity (top), a 5´ to 3´ polarity (middle), and a these enzymes play critical roles in polynucleotide exonuclease activity—which modifications inhibit bidirectional nuclease (bottom). repair, recycling, error correction, and protection nucleotide cleavage and which do not. from exogenous DNA (6-8). In vitro, exonucleases Recently, researchers at NEB have worked to are used in many applications where it is desirable characterize the interaction between exonucleases to remove certain nucleic acids. For example, for blocking nuclease activity, the phosphorothioate and modified polynucleotides, as part of a broader Exonuclease V (RecBCD) (Exo V, NEB #M0345) is bond (20-23), but will also discuss the use of other effort to gain deeper insight into the sequence and often used to remove contaminating linear ssDNA modifications to inhibit nuclease activity, as well structural determinants of nuclease activity and and dsDNA from plasmid preparations (4,9); T7 as which modifications have little to no effect on specificity. In an effort to catalog the modifications Exonuclease (T7 Exo, NEB #M0263) can be used exonuclease digestion. that inhibit exonuclease digestion, we have treated to generate 3´ overhangs in DNA (4, 10, 11); polynucleotides containing a range of modifications Exonuclease I (Exo I, NEB #M0293), Thermolabile Phosphorothioate linkages (including non-standard bases, sugars and backbone Exonuclease I (NEB #M0568) or Exonuclease VII chemistries) with exonucleases under the recom- A phosphorothioate (pt) bond is a phosphodiester (Exo VII, NEB #M0379) can be used to eliminate mended in vitro reaction conditions. This article will linkage where one of the two non-bridging ssDNA PCR primers, leaving double-stranded prod- summarize data from the literature, as well as the oxygens has been replaced by a sulfur (Figure 2). ucts undigested (12, 13), and Lambda Exonuclease key results from NEB’s work related to under- This modification has been used for decades to (Lambda Exo, NEB #M0262) can be used to con- standing the activity of exonucleases on chemically inhibit nuclease phosphodiesterase and phos- vert dsDNA to ssDNA for a variety of applications modified polynucleotides. We will focus on the phoryl transferase activities, as well as for gaining (14-16). More information on common applications most widely used—and most successful—method continued on page 2... 1 Figure 2: for example, Exo V, Exo VII and T5 Exonuclease Examples of common nucleotide modifications and their effect on exonuclease activity (T5 Exo, NEB #M0363) all can cleave, leaving short oligos instead of cutting at every bond in a Modied phosphodiesters (pt bonds) Modied bases do not inhibit series, and thus can digest DNA by skipping over block exonuclease activity exonuclease activity termini blocked by multiple pt bonds and cleaving -O O O 5´ – 5´ O OH at the first phosphodiester (5, 26). Importantly, any O– - O P O Base O O O P O enzyme with endonuclease activity, like DNase I, - Base O O O O O T O– O H O N will simply ignore the ends and degrade the O O P O HN N Base 5´ H O O S P O O– O Base O O N O O P O– polynucleotides from the inside out (unless every O– O O O O P – phosphodiester bond is replaced by a phospho- O O O 5´-Fluorescein dT O P – O O O 3´ O T OH 3´ rothioate). Keeping these important exceptions in 3´ HO mind, phosphorothioate bonds remain the most O NH2 O Phosphorothioate 3´-inverted dT 5´-inverted dT N N 5´ HN generally applicable (and relatively inexpensive) (pt) bond 5´ 5´ N N O NH2 O O N N O O N way to protect oligonucleotides from digestion O O H O by exonucleases. For a complete list of DNA O O O 5´ and 3´ end modication 3´ 3´ 3´ exonucleases and their interaction with pt bonds, does not block exonuclease activity IsoC IsoG Super T view our selection chart, Activity of Exonucleases O and Non-Specific Endonucleases, at go.neb.com/ HN NH O ExosEndos. H H Sugar modications inhibit H P N O O Base O– S O most exonuclease activity O 5´ 5´ 5´ 2´-modified nucleosides 5´ Biotin O O P O– O Base O Base O Base Generally, DNA exonucleases do not digest RNA O O O O 3´ O portions of oligonucleotides, though RNA is itself 5´ P O OH O OMe O O O O – Base O susceptible to RNases and nonspecific hydrolysis. O O 3´ 3´ 3´ OMe HN NH We have further found that hybridizing RNA to O 3´ Biotin H H – Ribo 2´-Methoxy 2´-Methoxyethyl O P O H DNA strands does not block the activity of dsDNA N (MOE) O S O exonucleases on the DNA strand. Hybridization of ssDNA to RNA will block the activity of ssDNA exonucleases as effectively as hybridization to mechanistic insights into these enzymes (20, 23). Phosphorothioates can block many, but not all, dsDNA. Additionally, certain 2´-O-modified Chemically, the substitution of oxygen with sulfur exonucleases. To block exonuclease cleavage, the riboses, are both stable to spontaneous hydrolysis does not dramatically change the reactivity of pt bonds must be placed at the end(s) where the and offer strong resistance to exonuclease activity the bond, and pt-containing polynucleotides can enzyme initiates, e.g., the 5´ end for Lambda Exo (27). 2´-O-methyl and 2´-O-methoxyethyl (MOE) still function in many enzymatic reactions. In a and the 3´ end for Exo III. It is important to note nucleosides, which contain bulky substituents off typical phosphodiester bond, the two non-bridging that a single pt bond is insufficient to fully protect the sugar ring, have been shown to grant strong oxygens are chemically equivalent. When one of an oligonucleotide from exonuclease digestion. resistance to nucleases and additionally increase the these oxygens is replaced by sulfur, however, the When the pt bond is installed via an oxidation step strength of annealing to complementary DNA and phosphorus is now connected to four distinct during phosphoramidite synthesis, a nearly equal RNA. These features have found utility in antisense groups, rendering it a chiral center with two amount of each isomer (SP and RP) is formed at each nuclease strategies, to make oligonucleotides that possible configurations referred to as “SP” and pt linkage (20). Since most enzymes can cleave one are both resistant to degradation and able to bind “RP” (Figure 3). It is this key feature that confers of these isomers, a single chemically installed pt will tightly to target RNAs.
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