Generation and Single-Molecule Characterization of a Sequence

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Generation and single-molecule characterization of a sequence-selective covalent cross-link mediated by mechlorethamine at a C-C mismatch in duplex DNA for discrimination of a disease-relevant single nucleotide polymorphism Ruicheng Shi, Maryam Imani Nejad, Li-Qun Gu, Xinyue Zhang, and Kent S. Gates Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00663 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Bioconjugate Chemistry

Generation and single-molecule characterization of a sequence-selective covalent cross-link mediated by mechlorethamine at a C-C mismatch in duplex DNA for discrimination of a disease-relevant single nucleotide polymorphism Ruicheng Shic,#, Maryam Imani Nejada,#, Xinyue Zhang,c Li-Qun Gu c,*, and Kent S. Gates a,b,* aUniversity

of Missouri

Department of Chemistry 125 Chemistry Building Columbia, MO 65211 bUniversity

of Missouri

Department of Biochemistry 125 Chemistry Building Columbia, MO 65211 cUniversity

of Missouri

Department of Bioengineering and Dalton Cardiovascular Research Center Columbia, MO 65211 * To whom correspondence should be addressed: email: [email protected]; phone: (573) 882-6763; FAX: (573) 882-2754; email: [email protected]; phone: (573) 882-2057 #These authors contributed equally to the work

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Abstract Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand but the reversible nature of nucleic acid hybridization presents an inherent challenge:

short probes that provide high target specificity have relatively

low target affinity resulting in signal losses.

Sequence-specific covalent

cross-linking reactions have the potential to provide both selective target capture and durable signal.

Here we explore a novel approach involving

sequence-specific

cross-linking

covalent

of

a

probe

to

target

DNA

combined with single-molecule nanopore detection of the cross-linked DNA. C-C

Here, we exploited the selective reaction of mechlorethamine at a mismatch

for

covalent

capture

of

a

target

DNA

sequence

corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the -hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the crosslinked

probe-target

complex.

Cross-linked

2


DNA

generates

an

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unmistakable deep and persistent current block (≥ 5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

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TOC Graphic

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INTRODUCTION The detection of disease-related and pathogenic DNA and RNA sequences is important in biotechnology and medicine.1-4 For example, widespread deployment of personalized medicine will require inexpensive and reliable assays for the detection of single nucleotide polymorphisms (SNPs).1,2,4-7 SNPs are the smallest differences that can exist in nucleic acid sequence but are very important in human health and disease.1,2,4-7

Not surprisingly, most sequence detection

strategies rely upon hybridization of a nucleobase-containing probe strand to the target nucleic acid, but the reversible nature of nucleic acid hybridization presents inherent challenges. 15-20

nucleotides)

provide

high

target

Short probes (approximately specificity

because

mismatch significantly destabilizes the probe-target complex.

a

single

However,

short probes are prone to denaturation (melting) during analysis, resulting in diminished signal intensity.8-13 Sequence-selective covalent cross-linking reactions can be used to stabilize probe-target complexes14-16 and also may provide an additional layer of target selectivity.17-24 A major challenge in this regard is the identification 5



of reactions that generate high cross-link yields in a programmable manner.

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For

example, we recently described the use of a probe containing an abasic site that sequence-selectively generates a covalent cross-link with an adenine residue on the opposing strand.17

This approach enables the detection of NA mutations and,

indirectly, NT mutations. Other cross-linking reactions must be employed for the detection of NC and NG mutations. Along these lines, we set out to explore the utility of a reaction involving cross-link formation by the anticancer agent mechlorethamine in DNA duplexes that contain C-C mismatches.25-27 We exploited this cross-linking reaction coupled with gel electrophoretic and single-molecule nanopore analysis of the resulting cross-linked probe-target complex for the detection of a cancerdriving TG mutation at position 1799 of the human BRAF kinase gene sequence.28-30 This mutation encodes a V600G substitution that makes the protein a target for the anticancer drugs vemurafenib and dabrafenib.28-30

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Figure 1

Figure 1. Covalent cross-linking of the probe-mutant BRAF gene sequence by mechlorethamine.

Probe-target duplexes with the non-

coding strands of mutant (1) and wild-type (2) BRAF gene sequences are shown above.

The probe strands were labeled on the 5’-end with

32P

in

the gel electrophoresis experiments. Structures of the cross-linking agent mechlorethamine and the mechlorethamine-derived C-C cross-link are shown below. RESULTS AND DISCUSSION Selective Detection of a Mutant BRAF Gene Sequence by Mechlorethaminemediated Cross-Linking of a C-C Mismatch in a Probe-Target Duplex. The TG mutation on the coding strand of the BRAF kinase gene can be analyzed using probes that detect the corresponding AC transversion on the complementary, non-coding

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strand. Accordingly, we designed a probe that would generate a C-C mismatch upon hybridization with the mutant sequence and a C-A mismatch when hybridized with the wild-type sequence (Figure 1).

We then examined whether the C-C duplex was

selectively cross-linked by mechlorethamine. Treatment of the 32P-labeled probe-mutant duplex 1 with mechlorethamine (100 M) in Tris buffer (10 mM, pH 7.3) containing NaCl (100 mM) and DMF (10% v/v) at 37 ºC for 4 h, followed by analysis using denaturing polyacrylamide gel electrophoresis gave 7.7  0.7% yield of a slowly migrating band on a denaturing polyacrylamide gel, consistent expected27,31-33 for a cross-linked duplex (Figure 2).

with

that

In contrast, no

detectable cross-link was generated in the wild-type probe-target duplex 2 (Figure 2, lanes 4 and 5).

Interestingly, the selectivity for cross-linking the

duplex containing the C-C mismatch was better in Tris buffer than in phosphate buffers (Figure S1). A time-course experiment revealed that cross-link formation in the probemutant duplex 3 by mechlorethamine was rapid, reaching a maximum yield within 5 h (Figure 3). The signal for cross-linked DNA was completely stable over the course of 10 days at room temperature (Figure S2). Iron-EDTA footprinting experiments31,32,34,35 established that the cross-link induced by mechlorethamine bridges the two mismatched C residues in duplex 1 (Figures S3 and S4).

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Figure 2

Figure 2. Mechlorethamine selectively cross-links the 1799 TG mutant probe-target duplex. Core sequences of duplexes generated by hybridization of the probe strand with mutant and wild-type sequences are shown above. Gel electrophoretic analysis of crosslink formation in the 21 bp duplexes shown in Figure 1. Lane 1: marker lane containing labeled probe strand alone; lane 2: mutant-probe duplex 1+mechlorethamine (Mec, 100 M), 2 h, 37 ˚C, (4.5% cross-link); lane 3: mutant-probe duplex 1+mechlorethamine (100 M), 24 h, 37 ˚C, (7.7% cross-link); lane 4: wild type-probe duplex 2+mechlorethamine (100 M), 2 h, 37 ˚C; lane 5: wild type-probe duplex 2+mechlorethamine (100 M), 24 h, 37 ˚C; lane 6: marker lane containing labeled probe strand alone.

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Figure 3

Figure 3.

Time course for mechlorethamine-induced cross-link formation

in the BRAF mutant probe-target duplex 3.

The probe strand was

labeled on the 5’-end with 32P and the cross-linking reaction carried out with mechlorethamine (100 M) in Tris buffer (10 mM, pH 7.3) containing NaCl (100 mM) and DMF (10% v/v) at 37 ºC. The labeled DNA was separated by electrophoresis using a 20% denaturing polyacrylamide gel and visualized by phosphorimager analysis. The yield of cross-link formation in duplex 3 at 4 h was 8.3 ± 0.3%. The

cross-linking

of

the

C-C

probe-target

duplex

1

by

mechlorethamine can be used to quantitatively measure the fraction of mutant

versus

wild-type

BRAF sequence in a sample.

Mixtures

containing various proportions of mutant and wild-type duplexes were denatured by warming the samples (50-70 ˚C) in the presence of the 32P-labeled

probe strand, followed by cooling and incubation at 37 ˚C

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with mechlorethamine (100 M, 4 h).

The extent of cross-link formation

was assessed by gel electrophoretic analysis.

Across the range of 3-

100% mutant duplex we observed a clear correlation between the fraction of mutant duplex and cross-link yield (Figure 4).

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Figure 4

Figure 4. Quantitative detection of the fraction of mutant BRAF sequence present in mixtures of mutant and wild-type duplexes. Samples containing various proportions of mutant and wild-type BRAF duplexes (21 bp) were denatured by warming at 70 °C in the presence of the

32P-labled

probe, cooled, and incubated at

37 °C with mechlorethamine (100 M) for 4 h.

The amount of cross-

linked duplex in each sample was assessed by gel electrophoretic analysis.

Single-Molecule Detection of the Cross-Linked Probe-Target Duplex Using the Hemolysin Nanopore. SNP detection benefits from sensitive methods that provide an unmistakable signal output when the target sequence is present. Nanopore devices have remarkable powers for the characterization of nucleic acid structures.36-41 Here, we employed the -hemolysin (-HL) protein nanopore device for singlemolecule detection of the cross-linked probe-mutant duplex 3. experiments, a single spanning

a

lipid

In these

ion channel provides a pore 1.4 nm in width42

bilayer

that

separates

12

two


chambers

of

aqueous

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electrolyte.43-45

Application of an electric potential across the bilayer

induces a readily measured ion current through the channel.

The

sequence and structure of various nucleic acids can be characterized based upon the changes in current flow (current signatures) produced when they are driven into (and usually through) the pore by the electrophoretic potential.36,46-51

We employed a probe strand with a dC30

overhang on the 3’-end (duplex 3, Figure 3) to increase the rates of duplex capture and unzipping in the -HL nanopore.49

Analysis of the

mixture generated by treatment of the probe-mutant duplex 3 with mechlorethamine revealed several distinct types of current signatures. Specifically, we observed 1) persistent current blocks consistent with capture and partial unzipping of the cross-linked DNA duplex (I/I0 = 11±1%), 2) short current blocks at the 100 ms scale consistent unzipping and translocation of the uncross-linked duplex (I/I0 = 14±1%), and 3) transient current blocks at the 100 s scale consistent with translocation of unhybridized single-stranded DNA through the pore (Figure 5).17,49

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The majority of the persistent blocks lasted until we reversed the voltage polarity of the device (after 10-20 s), causing the cross-linked duplex to “back out” of the channel.

When the voltage polarity was reset (green

arrows, Figure 5A and B), the reopened pore was again able to capture nucleic acid species in the mixture and record their current signatures. We have previously shown that the cross-link concentration can be measured

by

counting

event

frequencies

in

the

current

trace.17,49

Interestingly, a fraction (~15%) of the persistent current blocks gave way within 10-20 s to a period of near zero current flow (almost complete pore blockage, I/I0 = 2±1%), followed by a return to full (open-pore) current (Figure S5).

Current signatures of this type are consistent with

translocation of the cross-linked DNA through the -HL nanopore.49

14


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Figure 5

Figure 5. Single-molecule detection of the cross-linked probe-target duplex using the HL nanopore. Panel A: analysis of the probe-mutant duplex 3 treated with mechlorethamine (100 M) for 4 h at 37 ˚C at 120 mV in Tris-HCl (10 mM, pH 7.4) containing KCl (1 M) at 22 ± 1 ˚C. Three types of blocking events were observed in the cross-linking reaction mixture with mutant BRAF sequence: i. very short blocks associated with single-strand translocation events, ii. short blocks associated with duplex DNA unzipping and translocation events (black arrow) and, iii. persistent blocks generated by trapping of the cross-linked duplex in the -HL nanopore (red arrow). The majority of persistent blocks lasted until voltage polarity of the device was reversed (after 10-20 s), causing the cross-linked duplex to “back out” of the nanopore (green arrows).

When the voltage polarity was reset, the 15



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reopened pore was again able to capture nucleic acid species in the mixture. Panel B: analysis of the probe-wild type duplex treated with mechlorethamine. No persistent blocks were observed. Panel C: Expanded view of a persistent current block and proposed molecular events underlying this current signature. Panel D: Expanded of view of a short current block associated with capture, unzipping and translocation of an uncross-linked duplex and proposed molecular events underlying this current signature. Panel E: Expanded view of a very short current block associated with translocation of an unhybridized single stranded oligonucleotide and proposed molecular events underlying this current signature. The vertical axes in the current traces shown in panels D-E span from –50-150 pA.

Importantly, when the probe-wild type duplex 4 (Figure S6), was treated with mechlorethamine we did not observe persistent current blocks in the nanopore analysis (Figure 5B). showed

that

mismatched

neither analog

the 4

C-C

Further control experiments

mismatched

(untreated

persistent current blocks (Figure S5).

with

duplex

3

nor

mechlorethamine)

the

C-A

produced

The results show that nanopore

analysis provides a method for unambiguous detection of the cross-linked probe-target duplex 3 derived from the mutant BRAF gene sequence.

CONCLUSIONS We developed a system in which hybridization of a probe strand 16


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with a mutant BRAF gene sequence generates a C-C mismatch at the mutation site.

We used the anticancer drug mechlorethamine combined

with gel electrophoretic and nanopore analysis for the selective and unambiguous detection of the C-C mismatch in the probe-target duplex. Mechlorethamine

that

generates

aziridinium ions that alkylate various nucleobases in DNA.52-58

In typical

DNA

duplexes

is

lacking

a

bifunctional

5’-GNC

sites,

electrophile

micromolar

concentrations

mechlorethamine generate low yields of interstrand cross-links.54,58

of In

contrast, we find that mechlorethamine generates relatively high cross-link yields in the probe-mutant duplex 1 containing the C-C mismatch (Figures 1 and

2).

No cross-link formation was observed in the probe-wild type

duplex containing a C-A mismatch (Figures 1 and 2).

Our findings mesh

with work in different sequences showing that C-C mismatches were cross-linking hotspots for mechlorethamine, while C-A mismatches were not.25 The cross-linking reaction is fast (t1/2 < 2 h) and the resulting cross-linked duplex is stable for days (Figures 3 and S2). Our footprinting data (Figures S3 and S4) along

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with previous precedents25,56,59 suggest that the mechlorethamine-mediated cross-link spans the N3-positions of the mispaired cytosine residues in the probe-mutant duplex 1 as shown in Figure 1.

In duplex DNA, the N3-position of cytosine residues

typically is occluded by hydrogen bonding with the complementary guanine residue.60

The dynamic nature of the unpaired bases in the C-C

mismatch61-63 presumably allows mechlorethamine to access the N3position.

Regardless of the mechanisms that underlie selective covalent

cross-linking

by

mechlorethamine

at

C-C

mismatches,

the

evidence

indicates that this reagent provides a robust method for the recognition and detection of C-C mismatches in duplex DNA (for other methods, see refs:

64-70).

We found that the cross-linked C-C duplexes produce unmistakable signals in both gel electrophoretic and nanopore analyses.

In denaturing

gel electrophoretic analysis, the cross-linked DNA appears as a slowlymigrating band that is easily separated from uncross-linked DNA (Figure 2).

Nanopore analysis of the cross-linked DNA duplex in a device

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employing the -HL ion channel reveals long current blocks (5-10 s) that are easily distinguished from the short blocks typically associated with the translocation

of

single-stranded

DNA

and

uncross-linked

(microsecond and millisecond blocks, respectively).49

duplexes

Presumably, this

behavior is not limited to devices utilizing the -HL ion channel and it may be interesting to examine signals arising from cross-linked DNA in devices that utilize different apertures such as solid-state or aerolysin nanopores.71 Interestingly, approximately 15% of the events involving capture of the C-C cross-linked duplex 3 were followed within 15-20 s by a period of near zero current flow, followed by a return to full, open-pore current (Figure S5).

It is unlikely that the applied electrostatic potential provides

sufficient energy to induce breakage of covalent cross-link bonds in the nanopore; rather we propose that the cross-linked duplex is able to translocate through the nanopore.

The exceedingly tight fit of the two

cross-linked strands within the nominal 1.4 nm opening of the nanopore

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presumably explains the period of nearly complete current blockage (Figure S5).

The ability of the duplex containing a centrally-located C-C

cross-link to translocate through the -HL nanopore is distinct from the behavior of other cross-linked duplexes that have been examined.17,49 The C-C cross-link may induce distortion or partial melting of the duplex63 that provides conformational flexibility necessary to access compact structures that translocate through the nanopore.

It is interesting and

potentially useful to observe that current signatures in the -HL nanopore can distinguish interstrand DNA-DNA cross-links that differ in chemical structure.

Importantly however, these intriguing nuances in current

signatures of various cross-linked DNAs do not detract from the ability of the nanopore to produce an unmistakable, “high contrast” signal for the detection of cross-linked probe-mutant duplexes.

Overall, the methods

described here combine to yield a unique strategy for SNP detection via selective cross-linking of a C-C mismatch in duplex DNA.

EXPERIMENTAL PROCEDURES 20


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Materials and Methods. Reagents were purchased from the following suppliers and were of the highest purity available: oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Uracil DNA glycosylase (UDG), and T4 DNA polynucleotide kinase (T4 PNK) were from New England Biolabs (Ipswich, MA). [γ32P]-ATP

(6000 Ci/mmol) was purchased from PerkinElmer. C-18 Sep-Pak cartridges

were purchased from Waters (Milford, MA), and BS Poly prep columns were obtained from

BioRad

(Hercules,

CA).

Acrylamide/bis-acrylamide

19:1

(40%

Solution/Electrophoresis) was purchased from Fisher Scientific (Waltham, MA) and mechlorethamine hydrochloride from Sigma-Aldrich (St. Louis, MO). The compound 1,2-diphytanoyl-sn-glycero-3-phosphocholine used for lipid bilayer formation was from Avanti Polar Lipids (Alabaster, AL, USA) and was used without further purification. Quantification of radioactivity in polyacrylamide gels was carried out using a Personal Molecular Imager (BIORAD) with Quantity One software (v.4.6.5). DNA

Cross-Linking

by

Mechlorethamine.

Single-stranded

2’-

deoxyoligonucleotides were 5’-32P-labeled using standard procedures.72 Labeled DNA was annealed72 with its complementary strand to give the duplexes shown in Figure 1. In a typical cross-linking reaction, mechlorethamine was introduced to the reaction mixture as a freshly-prepared stock solution in DMF to give a mixture containing mechlorethamine (100 µM) and labeled DNA in Tris buffer (10 mM, pH 7.3) containing NaCl (100 mM) and DMF (10% v/v) that was incubated at 37 ºC for 4 h unless otherwise specified. The DNA was ethanol precipitated from the reaction mixture,72 resuspended in formamide loading buffer 72, loaded onto a 20% denaturing polyacrylamide gel, and the gel electrophoresed for 5 h at 1600 V. The amount of radiolabeled DNA in each band on

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the gel was measured by phosphorimager analysis. The time course for the formation of the cross-link was carried out by incubating a solution containing labeled DNA (approximately 100,000 cpm) and Tris buffer (10 mM, pH 7.3) containing NaCl (100 mM) and mechlorethamine (100 µM) at 37 ˚C. At specified time points, aliquots (3 µL) were removed and formamide loading dye was added followed by storage at –20 ºC until gel electrophoretic analysis as described above. Hydroxyl Radical Footprinting to Determine the Location of the Cross-Link Induced By Treatment of Duplex 1 by Mechlorethamine. We employed literature protocols to footprint the cross-link duplex 1.34 In separate experiments, each strand was 5’-labeled using standard procedure.72 Labeled DNA was annealed with the unlabelled complement and the double stranded DNA (~400,000 cpm) was incubated in Tris buffer (10 mM, pH 7.3) containing NaCl (100 mM) and mechlorethamine (100 µM) at 37 ºC for 24 h. The DNA was ethanol precipitated, suspended in formamide loading buffer and separated on a 2 mm thick 20% denaturing polyacrylamide gel. The cross-linked duplex band was located using X-ray film, the band cut out of the gel, and the gel slice crushed, and the gel pieces were vortex-mixed in elution buffer (NaCl, 200 mM; EDTA, 1 mM) at room temperature for at least 1 h. The mixture was filtered through a Poly-Prep column to remove gel fragments, the residue was ethanol precipitated, and redissolved in water and diluted with 2x oxidation buffer (10 µL of a solution composed of sodium phosphate, 20 mM, pH 7.2; NaCl, 20 mM sodium ascorbate, 2 mM; H2O2, 1 mM). To this mixture was added a solution of iron-EDTA (2 µL, EDTA, 70 mM; (NH4)2Fe(SO4)2•6H2O, 70 mM) to start the reaction, and the mixture vortexed briefly and incubated at room temperature for 5 min before addition of thiourea stop solution (10 µL of a 100 mM

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solution in water). Hydroxyl radical footprinting reactions, Maxam-Gilbert G reactions, and Maxam-Gilbert A+G reactions were performed on the labeled duplex to generate marker lanes.73 The resulting DNA fragments were analyzed using gel electrophoresis as described above. Electrophysiology Measurements. A membrane of 1,2-diphytanoyl-

sn-glycero-3-phosphocholine

was

formed

on

a

small

orifice

of

approximately 150 μm diameter in a Teflon partition that separates two identical Teflon chambers. Each chamber contained 2 mL of electrolyte solution (1 M KCl, 10 mM Tris-HCl, pH 7.4). Less than 1 μL of αhemolysin was added to the cis chamber with stirring, after which, a conductance increase indicated the formation of a single channel.

For

multichannel recording, 2 to 5 μL of α-hemolysin was added. The ionic current through the α-hemolysin protein nanopore was recorded by an Axopatch 200B amplifier (Molecular Devices Inc., Sunnyvale, CA), filtered with a built-in 4-pole low-pass Bessel Filter at 5 kHz, and finally acquired into the computer using a DigiData 1440A A/D converter (Molecular Devices) at a sampling rate of 20 kHz. All the data recording and

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acquisition including single channel, multichannel and persistent blocking recording of DNA cross-links were controlled through a Clampex program (Molecular Devices) and the analysis of nanopore current traces was performed using Clampfit software 10.4 (Molecular Devices).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem Effects of buffer on cross-link yield and selectivity; stability of the mechlorethamine cross-link in duplex 1; footprinting data; current signature for translocation of cross-linked duplex 3 through the -HL nanopore; results of control experiments with uncross-linked duplexes 3 and 4.

AUTHOR INFORMATION Corresponding Authors *E-mails: [email protected] and [email protected] ORCHID Kent S. Gates: 0000-0002-4218-7411 Li-qun Gu: 0000-0002-8710-6160 Author Contributions

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#Ruicheng Shi and Maryam Imani Nejad contributed equally to this work Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are grateful to the National Institutes of Health for support of this work (HG009338 to KSG and LQG, ES021007 to KSG, and GM114204 to LQG). REFERENCES (1)

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Generation and Single-Molecule Characterization of a Sequence

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