Engineering Specific Cross-Links in Nucleic
`Acids Using Glycol Linkers
`
`UNIT 5.3
`
`Simple glycol linkers can be used to cross-link nucleic acid sequences. In the most
`straightforward approach, such cross-links can be used in place of nucleotide sequences
`to bridge two domains of higher-order nucleic acid structures. Such linkers can also be
`viewed as tethers between two independently hybridizing nucleic acid sequences, or
`between a nucleic acid and some other ligand or reporter group. Although most any carbon
`chain can be employed to introduce cross-links in nucleic acids, the hydrophilic nature
`of the ethylene glycol chain gives it one particular advantage. Whereas simple carbon
`chains may tend to collapse on themselves as the result of the hydrophobic effect, the
`glycol chains’ alternating ethyl and oxygen ether subunits are more likely to be hydrated
`in aqueous solutions and thus maintain a more extended conformation, which permits
`them to easily bridge two different sites within the macromolecule. Additionally, a variety
`of ethylene glycol–based linkers are readily available (Fig. 5.3.1) and only require simple
`protection reactions in order to be used as cross-linking agents.
`
`Oligo(ethylene glycol) linkers have been used most commonly to replace a portion
`(Williams and Hall, 1996) or the entirety of the loop structure at the end of DNA (Durand
`et al., 1990; Altmann et al., 1995) or RNA helices (Benseler et al., 1993; Ma et al., 1993;
`Thomson et al., 1993; Fu et al., 1994; Hendry et al., 1994; Komatsu et al., 1996),
`essentially to achieve cross-linking of the terminal residues of the double-stranded helix.
`However, in some cases ethylene glycol linkers have been used to tether different strands
`of nucleic acids (Cload and Schepartz, 1991; Amaratunga and Lohman, 1993; Moses and
`Schepartz, 1996) or even to tether minor groove-binding ligands to the nucleic acid
`(Robles et al., 1996; Rajur et al., 1997; Robles and McLaughlin, 1997). In most cases,
`the glycol linker is incorporated as part of the nucleic acid backbone, such that at each
`terminus the linker is incorporated into a phosphodiester linkage that also incorporates
`either the 3′ or 5′ hydroxyl of the adjacent nucleoside residue. It is also possible to
`incorporate more than a single linker at the same site. Thus, two residues of tri(ethylene
`glycol) could be used instead of hexa(ethylene glycol) (Benseler et al., 1993; Fu et al.,
`1994)—in the former case a negatively charged phosphodiester would bridge the two
`linkers. This approach can be used to generate structures with varying linker lengths via
`the preparation of only a single linker building block.
`In the most common protocol, the linker is protected at one terminus as the 4,4′-di-
`methoxytrityl derivative (see Basic Protocol 1), and is converted to a phosphoramidite at
`the second terminus (see Basic Protocol 2). With such derivatives, the linker is simply
`incorporated into the DNA or RNA sequence by the same procedures as are used for
`common nucleoside phosphoramidites (see Basic Protocol 3). Preparation of the pro-
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`Figure 5.3.1 Varying lengths for readily available ethylene glycol–based linkers.
`
`Contributed by Timothy O’Dea and Larry W. McLaughlin
`Current Protocols in Nucleic Acid Chemistry (2000) 5.3.1-5.3.8
`Copyright © 2000 by John Wiley & Sons, Inc.
`
`Methods for
`Cross-Linking
`Nucleic Acids
`5.3.1
`
`Oxford, Exh. 1008, p. 1
`
`

`

`BASIC
`PROTOCOL 1
`
`tected linker-phosphoramidites follows a common procedure regardless of length; proto-
`cols for the hexa(ethylene glycol) linker are presented here.
`
`PROTECTION OF THE GLYCOL CHAIN WITH A TRITYL GROUP
`The following protocol outlines the protection of one terminus of an ethylene glycol chain
`with a trityl group. The first reaction, illustrated in Figure 5.3.2, promotes monoprotection
`of the ethylene glycol chain with 4,4′-dimethoxytrityl chloride. Although the specific
`protocol for hexa(ethylene glycol) follows, this protocol has also been successful with
`glycol chains of various lengths: 1,3-propanediol, tri(ethylene glycol), the tetra- and
`penta-compounds, and so on. The monoprotected ethylene glycol product can be purified
`by silica-gel column chromatography.
`
`Materials
`Hexa(ethylene glycol) (HEG)
`Anhydrous pyridine (preferably freshly distilled)
`Nitrogen or argon gas
`4,4′-Dimethoxytrityl chloride (DMT-Cl)
`5% (v/v) methanol in dichloromethane
`10% (v/v) aqueous sulfuric acid (H2SO4; Table A.2A.1)
`Triethylamine (Et3N, TEA)
`Dichloromethane (CH2Cl2, DCM; preferably freshly distilled)
`5% (w/v) aqueous sodium hydrogen carbonate (NaHCO3)
`Sodium sulfate (Na2SO4)
`Methanol (CH3OH, MeOH)
`
`HO
`
`O
`
`O
`
`O
`
`O
`
`O
`
`OH
`
`OCH3
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`OH
`
`OCH3
`
`OCH3
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`P OCH2CH2CN
`N(i-Pr)2
`
`OCH3
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`G G
`
`CC
`
`C C G G
`
`Figure 5.3.2 Reaction pathway for the preparation of a glycol linker and a sample nucleic acid
`sequence containing the linker.
`
`Current Protocols in Nucleic Acid Chemistry
`
`Engineering
`Specific
`Cross-Links in
`Nucleic Acids
`Using Glycol
`Linkers
`5.3.2
`
`Oxford, Exh. 1008, p. 2
`
`

`

`Non-acid-generating desiccant: e.g., sodium hydroxide or calcium carbonate
`100-mL round-bottom flask and rubber stopper
`Device for maintaining nitrogen or argon atmosphere (e.g., balloon, syringe, and
`rubber stopper; see step 2)
`Needle and syringe
`Separatory funnel
`Silica gel
`Column for chromatography
`Rotary evaporator
`Thin-layer chromatography (TLC) apparatus (see APPENDIX 3D)
`
`CAUTION: Pyridine and its vapors are toxic; exposure to pyridine must be minimal. The
`reaction should be performed in a fume hood.
`
`Monoprotect ethylene glycol
`1. Coevaporate 1.25 g (5 eq, 4.43 mmol) HEG twice with ∼10 mL anhydrous pyridine
`in a 100-mL flask.
`
`2. Under an anhydrous nitrogen or argon atmosphere, add 10 mL anhydrous pyridine
`and a dry stir bar, and seal the 100 mL-flask with a rubber stopper.
`
`The easiest means to create a nitrogen or argon atmosphere is via a balloon sealed to a
`syringe with a needle. To construct: Remove plunger from syringe, cut off the now opened
`end, slip a balloon onto this end, and seal well with parafilm. Fill balloon with gas, attach
`needle, and punch needle through rubber stopper.
`
`3. Begin stirring at ambient temperature.
`
`4. In a separate flask under nitrogen, dissolve 300 mg (1 eq, 0.885 mmol) DMT-Cl in
`∼3 mL anhydrous pyridine.
`5. Using a syringe, puncture the rubber stopper and gradually add the DMT-Cl solution
`to the reaction flask.
`
`Useful increments are 0.5 mL every 5 min over a 30-min period.
`
`The reaction can be monitored by TLC (silica gel, 60 Å, see APPENDIX 3D) using 5%
`methanol in DCM as eluant. The Rf is 0.45. The product is visible under UV and turns
`orange when reacted with 10% aqueous H2SO4.
`6. After 2 hr, add 2 mL TEA and dilute with ∼25 mL DCM.
`TEA neutralizes the acid that has been generated, which otherwise will cleave the
`mono-DMT derivative of the ethylene glycol linker.
`7. Extract the organic layer twice with 5% NaHCO3 (∼40 mL) and once with distilled
`water (∼40 mL) using a separatory funnel.
`8. Dry the organic layer over Na2SO4 and remove solvent with a rotary evaporator.
`
`The product remains as a clear or slightly colored oil.
`
`Purify mono-DMT–ethylene glycol product
`9. Pack a silica-gel column (~15 g, roughly 10× expected solute amount), using 0.5%
`TEA in DCM as eluant.
`
`Again, TEA reduces the acidic nature of the silica gel, thus reducing decomposition of the
`mono-DMT–ethylene glycol during chromatography.
`
`10. Dissolve the mono-DMT–ethylene glycol product (from step 8) in a minimum
`quantity of DCM/TEA and pour onto the column. Elute with at least 400 mL of 0.5%
`
`Current Protocols in Nucleic Acid Chemistry
`
`Methods for
`Cross-Linking
`Nucleic Acids
`5.3.3
`
`Oxford, Exh. 1008, p. 3
`
`

`

`TEA in DCM, followed by a step gradient using 400-mL aliquots of 0.5% TEA/DCM
`containing from 0.5% to 3% MeOH.
`
`The product will elute in <3% MeOH.
`
`11. Test fractions by TLC (APPENDIX 3D; Rf = 0.45) using 5% MeOH in DCM as the eluant.
`12. Combine fractions containing the correct product and remove solvent by rotary
`evaporation (high vacuum is needed to remove excess TEA in product).
`
`13. Store in a sealed vial at ambient temperature over a desiccant.
`The 4,4′-dimethoxytrityl-protected hexa(ethylene glycol) product (DMT-HEG) is stable for
`several months with minimal decomposition provided it is not stored over a desiccant that
`liberates acid (e.g., P2O5).
`
`PHOSPHITYLATION OF THE MONOPROTECTED GLYCOL LINKER
`The following protocol details the phosphitylation of a 4,4′-dimethoxytrityl-protected
`glycol linker with 2-(cyanoethyl)-N,N-diisopropylchlorophosphoramidite. For an effi-
`cient reaction with high yield, conditions must be kept scrupulously anhydrous. While
`the following procedure outlines the use of a monoprotected hexa(ethylene glycol) linker,
`the protocol has been successful with monoprotected glycol compounds of various
`lengths. The reaction is illustrated in Figure 5.3.2.
`
`Materials
`4,4′-Dimethoxytrityl-protected hexa(ethylene glycol) (DMT-HEG; see Basic
`Protocol 1)
`Anhydrous pyridine (preferably freshly distilled; UNIT 3.2)
`Non-acid-generating desiccant: e.g., sodium hydroxide or calcium carbonate
`Nitrogen or argon gas
`Anhydrous dichloromethane (CH2Cl2, DCM; preferably freshly distilled)
`Diisopropylethylamine
`2-(Cyanoethyl)-N,N-diisopropylchlorophosphoramidite
`Ethyl acetate
`10% (v/v) triethylamine (Et3N, TEA) in ethyl acetate
`5% (w/v) aqueous NaHCO3
`Saturated aqueous NaCl
`Sodium sulfate (Na2SO4)
`25-mL round-bottom flask and rubber stopper
`
`CAUTION: Pyridine and its vapors are toxic; exposure to pyridine must be minimal. The
`reaction should be performed in a fume hood.
`
`Phosphitylate DMT–ethylene glycol
`1. Coevaporate 300 mg (1 eq, 0.51 mmol) of DMT-HEG twice with ∼10 mL anhydrous
`pyridine. Place under high vacuum over a non-acid-generating desiccant and leave
`overnight.
`
`2. In a rubber-stoppered 25-mL round-bottom flask with a dry stir bar under an
`anhydrous nitrogen or argon atmosphere, dissolve the DMT-HEG in 1 mL anhydrous
`DCM and 0.22 mL (3 eq, 1.54 mmol, 157 mg) anhydrous diisopropylethylamine.
`
`A balloon sealed to a syringe provides an easy means to create a nitrogen or argon
`atmosphere (see Basic Protocol 1, step 2, for details).
`
`TEA may be used here as an alternative to diisopropylamine, if preferred.
`
`Current Protocols in Nucleic Acid Chemistry
`
`BASIC
`PROTOCOL 2
`
`Engineering
`Specific
`Cross-Links in
`Nucleic Acids
`Using Glycol
`Linkers
`5.3.4
`
`Oxford, Exh. 1008, p. 4
`
`

`

`3. While stirring, add 0.115 mL (1 eq, 0.51 mmol, 121 mg) 2-(cyanoethyl)-N,N-diiso-
`propylchlorophosphoramidite to the reaction flask using a syringe.
`
`The reaction can be monitored via TLC using 9:1 (v/v) ethyl acetate/TEA as the eluant.
`The Rf of DMT-HEG is 0.50 and that of DMT-HEG-phosphoramidite is 0.80. The product
`is visible under UV and turns orange when treated with 10% H2SO4.
`4. After 25 min, dilute reaction with ∼20 mL ethyl acetate.
`
`5. Extract the organic layer twice with 5% aqueous NaHCO3 and once with saturated
`aqueous NaCl.
`
`6. Filter organic layer over Na2SO4 and evaporate solvent with rotary evaporator.
`
`Purify DMT–ethylene glycol–phosporamidite
`7. Pack a silica-gel TLC column with 1% (v/v) TEA in ethyl acetate.
`
`8. Elute the product with increasing percentages of TEA (1% to 5%) in ethyl acetate.
`
`9. Test fractions by TLC (Rf = 0.80) using 10% TEA in ethyl acetate as the eluant.
`
`10. Combine fractions containing the correct product and remove solvent using rotary
`evaporator (high vacuum is needed to remove the TEA).
`11. Store in a sealed vial at −20°C
`The DMT-HEG-P will remain stable for several weeks.
`
`PREPARATION OF ETHYLENE GLYCOL LINKERS FOR
`INCORPORATION INTO OLIGONUCLEOTIDES
`
`BASIC
`PROTOCOL 3
`
`The DMT-protected and phosphitylated glycol linkers can be inserted into DNA se-
`quences using standard automated phosphoramidite synthesis. Since the glycol linker is
`an oil, several preparative steps facilitate its incorporation using an automated synthesizer.
`
`For an overview of oligonucleotide synthesis, see APPENDIX 3C.
`
`Materials
`Dimethoxytrityl-protected hexa(ethylene glycol) phosphoramidite (DMT-HEG-P)
`(see Basic Protocol 2)
`Anhydrous dichloromethane (CH2Cl2, DCM; preferably freshly distilled)
`Anhydrous acetonitrile (preferably freshly distilled)
`Bottle from DNA synthesizer, tared
`
`1. Dissolve 266 mg DMT-HEG-P (0.34 mmol) in 1.00 mL anhydrous CH2Cl2 under an
`anhydrous nitrogen or argon atmosphere.
`
`A balloon sealed to a syringe provides an easy means to create a nitrogen or argon
`atmosphere (see Basic Protocol 1, step 2, for details).
`
`2. Using a syringe, transfer 0.100 mL DMT-HEG-P solution to a suitable tared DNA
`synthesizer bottle.
`
`3. Remove solvent on rotary evaporator and dry under high vacuum overnight.
`
`4. Weigh DNA synthesis bottle to determine exact amount of DMT-HEG-P.
`5. Dissolve 24 mg DMT-HEG-P (∼30 µmol) in 250 µL anhydrous acetonitrile.
`Care must be taken to ensure DMT-HEG-P is dissolved completely.
`
`Current Protocols in Nucleic Acid Chemistry
`
`Methods for
`Cross-Linking
`Nucleic Acids
`5.3.5
`
`Oxford, Exh. 1008, p. 5
`
`

`

`6. Place the bottle on the automated DNA synthesizer and purge as recommended by
`the manufacturer.
`
`7. The ethylene glycol linker can now be incorporated into oligonucleotides by solid-
`support synthesis using standard phosphoramidite protocols.
`
`The oligonucleotide can be synthesized with the DMT group on and then purified by HPLC
`analysis, or with the DMT group off and then purified by gel electrophoresis.
`
`If poor coupling occurs, see Critical Parameters for possible solutions.
`
`COMMENTARY
`Background Information
`The cross-linking agents described in this
`unit are those that are employed during the
`assembly of the DNA sequences—in this re-
`spect they are introduced at very specific sites.
`Other simple carbon-based linkers can also be
`employed in a similar manner, but as noted
`earlier, simple carbon chains may tend to col-
`lapse on themselves in an aqueous environ-
`ment, while the glycol chains are more likely
`to be hydrated and thus maintain a more ex-
`tended conformation. Other approaches to
`cross-linking are also available, most notably
`the introduction of thiol-based linkers, which
`upon oxidation form a disulfide cross-link be-
`tween two sites within a higher-order nucleic
`acid complex (Ferentz and Verdine, 1991;
`Wolfe and Verdine, 1993; Goodwin and Glick,
`1994; Cain and Glick, 1998; UNITS 5.1 & 5.4).
`It has been difficult to design effective pro-
`tocols to confirm the presence of the linker
`within the nucleic acid sequence. With other
`types of modified sequences, DNA digests can
`often be used to confirm the presence of the
`modification. In the present case, the linkers are
`not easily identifiable, and such digests only
`confirm the presence of the nucleoside compo-
`nents. However, when the linker is present in
`RNA, it is possible to treat a small quantity of
`the nucleic acid fragment with T2 RNase and
`an alkaline phosphatase, which results in cleav-
`age of all linkages save that between the linker
`and the 5′ terminus of the nucleotide (there is
`no requisite 2′-OH at this linkage). The result-
`ing nucleoside attached to the linker can then
`be identified after the appropriate standard is
`prepared (Fu et al., 1994).
`Careful analysis of the digest by HPLC, with
`the use of an appropriate standard, can confirm
`the presence of the linker in the sequence of
`interest (Fu et al., 1994). However, this procedure
`can be tedious, and requires the preparation of the
`necessary standard(s). Recent work in the
`authors’ lab (D.J. Fu, G. Xiang, and L. McLaugh-
`lin, unpub. observ.) has indicated that MALDI-
`
`TOF (UNIT 10.1) analyses of such nucleic acid
`analogues are much simpler and are effective in
`providing evidence for the presence of the linker
`in both DNA and RNA target sequences.
`
`Critical Parameters
`The synthesis of these linkers should not
`present significant problems for anyone with
`even a moderate level of laboratory experience.
`As can be noted with the protocol, the glycol
`linkers tend to be quite inexpensive and are used
`in excess over the DMT-Cl reagent to ensure
`that only the mono-protected product results.
`These reactions can be performed with
`stoichiometries of 1:1, but usually some of the
`bis-protected DMT-linker results. This un-
`wanted product can be removed during the
`purification step.
`For these protocols to succeed, the reactions
`must be performed under anhydrous condi-
`tions. Because of DMT-Cl’s sensitivity to
`water, great care must be taken to maintain a
`dry environment for the reaction. All ethylene
`glycol reagents should be coevaporated with
`pyridine and kept under vacuum before use. A
`nitrogen or argon atmosphere for the reaction
`helps maintain the anhydrous conditions while
`the reaction is being run. The simplest appara-
`tus is a balloon fixed to a syringe body and filled
`with dry argon/nitrogen. The reaction flask is
`sealed with a rubber septum and a needle af-
`fixed to the syringe is pushed through the sep-
`tum. This simple apparatus keeps the reaction
`mixture under a slight positive pressure with an
`anhydrous inert gas.
`High yields require that one be aware of the
`acid lability of the DMT-protecting group. To
`limit decomposition as a result of trace quanti-
`ties of acid, a small amount of the organic base
`triethylamine (TEA) is added during work-up.
`It is also critical to have some TEA present
`(∼0.5%) during the chromatographic purifica-
`tion step employing a silica-gel column. TEA
`neutralizes the slight acidity of the silicic acid,
`promoting greater stability of the product when
`
`Current Protocols in Nucleic Acid Chemistry
`
`Engineering
`Specific
`Cross-Links in
`Nucleic Acids
`Using Glycol
`Linkers
`5.3.6
`
`Oxford, Exh. 1008, p. 6
`
`

`

`it is adsorbed on the column support. The
`∼0.5% TEA does not significantly alter chro-
`matographic mobility, and its presence results
`in a greater overall yield of recovered product.
`For the phosphitylation protocol, taking
`into consideration the lability of the phosphor-
`amidite is critical for a successful experi-
`ment. Once synthesized, effort must be taken
`to minimize the exposure of the phosphoram-
`idite to air and acidic compounds of any kind.
`Both the reagent and the isolated product
`should be stored in parafilm-sealed vials at
`−20°C. When the phosphoramidite reagent
`loses its pale-yellow color and becomes a
`deeper yellowish-orange color, the reagent
`has typically degraded and should not be
`used.
`Preparation of the linker for use with a DNA
`synthesizer is only complicated by the fact that
`it is an oil rather than a solid. The authors have
`found the simplest procedure is one in which
`some of the oil is transferred to a suitable flask,
`weighed, and dissolved in sufficient anhydrous
`solvent. From this solution, an aliquot corre-
`sponding to ∼30 µmol of linker (per coupling)
`is transferred to the DNA synthesis bottle. The
`solvent is then removed from the bottle under
`vacuum and the residue is kept under high
`vacuum overnight. The requisite amount of
`acetonitrile can then be added to the bottle
`before the latter is attached to the DNA synthe-
`sis machine—ensure that the residue in the
`bottle completely dissolves first.
`To obtain efficient coupling of the linker to a
`DNA strand, the synthesizer programming need
`not to be altered. However, if efficient coupling
`is not achieved, several parameters can be
`changed to attain better coupling. First, extended
`“wait” periods can be added to the cycle—these
`are typically the time periods during which the
`coupling reaction takes place. A second option is
`to perform two coupling steps in sequence with-
`out any intervening capping or oxidation steps.
`
`Anticipated Results
`Yields for the protection of the glycol linker
`with DMT should be >70%. When isolated by
`column chromatography the DMT-ethylene
`glycol product is a pale-orange oil with Rf =
`0.45 (5% MeOH in DCM). The yields expected
`for the phosphitylation protocol should be
`>70% when isolated from the column as a clear
`oil (Rf = 0.8, 1:9 TEA/ethyl acetate). Successful
`phosphitylation can be achieved without the
`column chromatography step. In this case, sim-
`ply perform the aqueous work-up, dry the so-
`lution, and evaporate to an oil. 31P NMR will
`
`Current Protocols in Nucleic Acid Chemistry
`
`confirm the ratio of the phosphitylated product
`to any phosphorus contaminants. So long as the
`latter are minimal in quantity, effective incor-
`poration of the linker can be obtained with
`material prepared in this manner.
`
`Time Considerations
`Monoprotection of ethylene glycol and its
`isolation can be accomplished in <5 hr. The
`phosphitylation protocol can be done in <2 hr
`when the DMT-ethylene glycol product is pre-
`pared ahead of time (see Basic Protocol 1).
`Incorporation of the ethylene glycol linker into
`the oligonucleotide will not require more than
`a half-hour beyond the normal coupling time
`required of a standard phosphoramidite.
`
`LITERATURE CITED
`Altmann, S., Labhardt, A.M., Bur, D., Lehmann, C.,
`Bannwarth, W., Billeter, M., Wuthrich, K., and
`Leupin, W. 1995. NMR studies of DNA duplexes
`singly cross-linked by different synthetic linkers.
`Nucl. Acids Res. 23:4827-4835.
`Amaratunga, M. and Lohman, T.M. 1993. Es-
`cherichia coli Rep helicase unwinds DNA by an
`active mechanism. Biochemistry 32:6815-6820.
`Benseler, F., Fu, D.J., Ludwig, J., and McLaughlin,
`L.W. 1993. Hammerhead-like molecules con-
`taining non-nucleoside linkers are active RNA
`catalysts. J. Am. Chem. Soc. 115:8483-8484.
`Cain, R.J. and Glick, G.D. 1998. Use of cross-links
`to study the conformational dynamics of triplex
`DNA. Biochemistry 37:1456-1464.
`Cload, S.T. and Schepartz, A. 1991. Polyether teth-
`ered oligonucleotide probes. J. Am. Chem. Soc.
`113:6324-6326.
`Durand, M., Chevrie, K., Chassignol, M., and
`Thuong, N.T. 1990. Circular dichroism studies
`of an oligodeoxyribonucleotide containing a
`hairpin loop made of a hexaethylene glycol
`chain—conformation and stability. Nucl. Acids
`Res. 18:6353-6359.
`Ferentz, A.E. and Verdine, G.L. 1991. Disulfide
`cross-linked oligonucleotides. J. Am. Chem. Soc.
`113:4000-4002.
`Fu, D.J., Benseler, F., and McLaughlin, L.W. 1994.
`Hammerhead ribozymes containing non-nucleo-
`side linkers are active RNA catalysts. J. Am.
`Chem. Soc. 116:4591-4598.
`Goodwin, J.T. and Glick, G.D. 1994. Synthesis of a
`disulfide stabilized RNA hairpin. Tetrahedron
`Lett. 35:1647-1650.
`Hendry, P., Moghaddam, M.J., McCall, M.J., Jen-
`nings, P.A., Ebel, S., and Brown, T. 1994. Using
`linkers to investigate the spatial separation of the
`conserved nucleotides A9 and G12 in the ham-
`merhead ribozyme. Biochim. Biophys. Acta
`1219:405-412.
`Komatsu, Y., Kanzaki, I., and Ohtsuka, E. 1996.
`Enhanced folding of hairpin ribozymes with re-
`placed domains. Biochemistry 35:9815-9820.
`
`Methods for
`Cross-Linking
`Nucleic Acids
`5.3.7
`
`Oxford, Exh. 1008, p. 7
`
`

`

`Ma, M.Y.X., McCallum, K., Climie, S.C., Kuper-
`man, R., Lin, W.C., Sumner-Smith, M., and
`Barnett, R.W. 1993. Design and synthesis of
`RNA miniduplexes via a synthetic linker ap-
`proach. 2. Generation of covalently closed, dou-
`ble-stranded cyclic HIV-1 TAR RNA analogs
`with high Tat-binding affinity. Nucl. Acids Res.
`21:2585-9.
`Moses, A.C., and Schepartz, A. 1996. Triplex teth-
`ered oligonucleotide probes. J. Am. Chem. Soc.
`118:10896-10897.
`Rajur, S.B., Robles, J., Wiederholt, K., Kuimelis,
`R.W., and McLaughlin, L.W. 1997. Hoechst
`33258 tethered by a hexa(ethylene glycol) linker
`to the 5′-termini of oligodeoxynucleotide 15-
`mers: Duplex stabilization and fluorescence
`properties. J. Org. Chem. 62:523-529.
`Robles, J. and McLaughlin, L.W. 1997. DNA triplex
`stabilization using a tethered minor-groove bind-
`ing Hoechst 33258 analogue. J. Am. Chem. Soc.
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`
`Contributed by Timothy O’Dea and
` Larry W. McLaughlin
`Boston College
`Chestnut Hill, Massachusetts
`
`Engineering
`Specific
`Cross-Links in
`Nucleic Acids
`Using Glycol
`Linkers
`5.3.8
`
`Current Protocols in Nucleic Acid Chemistry
`
`Oxford, Exh. 1008, p. 8
`
`